Toy Story 3 masih bercerita tentang sekumpulan mainan anak-anak, si koboy Woody dan kawan-kawan. Kali ini Woody dan kawan-kawannya terjebak dalam sebuah dilema, diam menjadi penghuni loteng demi pengabdian kepada Andy, sang pemilik, atau pergi mencari pemilik baru agar terus bisa menghibur anak-anak lain.

Andy yang mulai beranjak dewasa tentu saja mulai jarang memainkan Woody dan teman-temannya. Setelah diterima di perguruan tinggi, Andy memutuskan hanya Woody yang akan pergi menemaninya, sementara yang lainnya akan disimpan menjadi penghuni loteng. Karena suatu ketidaksengajaan, Ibunya mengira, Woody akan menyumbangkan mainan bekas tersebut ke tempat penitipan anak, Sunnyside.

Dari ketidaksengajaan tersebut, kisah petualangan Woody Cs dimulai. Woody tak sependapat dengan teman-temannya untuk tinggal di tempat penitipan anak tersebut. Baginya, terus mengabdi pada Andy, sebagai pemilik adalah tugas utama mereka sebagai mainan. Namun dipimpin oleh Buzz, teman-temannya berpendapat lain, Andy tak lagi membutuhkan kehadiran mereka dan kini saatnyalah bagi mereka untuk mencari pemilik lain.

Lalu kecerobohan dari anak-anak pra-sekolah membuat Woody berpisah dengan teman-temannya. Woody berusaha untuk menyelamatkan teman-temannya dan menemukan diri mereka di rumah baru, tetapi problem muncul karena Buzz tiba-tiba rusak sewaktu berusaha melarikan diri. Woddy mencoba untuk me-reset Buzz, tetapi akhirnya malah membuat dia kembali ke versi spanyol.

Andy Davis, now 17, is a mere three days away from heading off to college and the toys are worried about their uncertain future. The toys haven't been played with in years and are desperate for attention from Andy. Most of the toys have been thrown out or have run away including Weezy, Etch-a-Sketch, the Army men and Little Bo Beep. Andy only allowed Sheriff Woody to go with him, while all the others were put into a trash bag headed for the attic, but Andy's mom thinks that the bag is full of garbage, and thus throws the toys away. The Toys manage to escape from their trash bag on the street and make it to the garage. The toys (unaware that they were intended to put in the attic) feel betrayed and decide to donate themselves to Sunnyside Daycare. Woody tries to explain to them the misunderstanding but before he could dissuade them, the car is started and on it way to Sunnyside, trapping Woody with them. The gang arrives at Sunnyside just as the children leave for recess, the Sunnyside toys welcome Andy's toys with open arms, including the kindly leader of the daycare, Lots-o'-Huggin' Bear (A.K.A. "Lotso"), a smooth-talking Ken doll, who falls in love with Molly's Barbie doll and a doll covered in crayon tattoos called Big Baby. The toys are keen on starting a new life at the daycare, except for Woody, who has suspicions about the daycare because of the Chatter Telephone and also thinks that the toys shouldn't turn their back on Andy so quickly.

Meanwhile, Woody reluctantly leaves to find Andy, and ended up getting stuck in a tree and taken by Bonnie, to meet her own toys, Chuckles the Clown, Mr. Pricklepants, Trixie, Buttercup, Dolly, the Peas-in-a-Pod and Totoro. As Woody tries to recuperate himself and continue his search for Andy, Chuckles explains to Woody the dangers of Sunnyside. Chuckles tells Woody that he, Lotso and Big Baby were once owned by a loving girl named Daisy. However, one day, she accidentally left them in the countryside. They returned to Daisy's house to find that Daisy's parents bought a new Lots-o'-Huggin' Bear for her, leaving Lotso feeling betrayed and hostile towards all toy owners. The toys stumble upon Sunnyside Daycare where Lotso and Big Baby quickly rose to power and transformed the daycare into a toy prison for those left there, while Chuckles was taken home by Bonnie. Woody realizes he must rescue his friends from Lotso and Big Baby.

Meanwhile, at the daycare, Buzz and the gang are "assigned" to the "caterpillar room" where they are played by very young kids too roughly. While the doors are locked at night, Buzz begins to jump over the window and finds Ken and other toys heading to the teacher lounge's snack machine. Buzz is tied to the "timeout chair" and is about to be tortured until Lotso arrives and orders the other toys to release him. Buzz asks Lotso if he and his friends can be transferred to the "butterfly room", but Lotso agrees to only allow Buzz to transfer. Buzz insists that he must stay together with his friends. Lotso, tired of him, orders to tie him again and, following the instruction manual of a Buzz Lightyear, resets Buzz and kidnaps the gang.

Woody returns to Sunnyside to rescue his friends and get back to Andy's house before he leaves for college the following day. Asking for help, Chatter Telephone informs Woody that he must get rid of a monkey toy that controls the AV room and watches everything that happens in the daycare through cameras. Woody and Slinky fight the monkey and get the keys to all the rooms. The gang later attempts to escape, but when Lotso finds them, Ken speaks up and convinces everyone that it was Lotso who turned Sunnyside into a prison instead of a joyful place for toys. Lotso gets mad at him and Woody speaks up and tells Big Baby that he was never replaced; it was only Lotso who was replaced. Big Baby is enraged by his trickery and throws Lotso in the dumpster. Everybody is about to escape but Woody is pulled into the dumpster by Lotso in the process. The rest of the gang try to save Woody but ends up getting thrown into the garbage truck heading to the landfill. They arrive at the landfill and help get Lotso unstuck from the conveyor belt, but Lotso later betrays them rather than halt the conveyor and leaves them to burn like the trash he thinks they are. Seeing no way out, the toys hold hands and prepare to meet their fate, but at the last minute, they're saved by a giant claw being operated by the Little Green Aliens. Hamm and Slinky want to teach Lotso a lesson for trying to kill them, but Woody convinces them that he's been enough trouble for them and Lotso faces retribution on his own when a garbage man (revealed to be a now grown Sid from the first film), who once had a Lotso of his own, finds him and ties him to the front of his truck with a few other mangled toys.

Woody, Buzz and the gang find the original garbage truck and they race to get home before Andy leaves. They all get to their boxes, but Woody realizes he wants a better life for his friends than in the attic, so he writes Bonnie's address on a Post-it and leaves it for Andy to see. Andy, instead of taking his beloved toys to the attic, leaves them to Bonnie, for her to take care of and love them. Woody has also stowed away in the box and Andy is at first doubtful and reluctant to leave him with Bonnie but finally relents, ending with Bonnie playing with Andy and her new toys, as well as Andy finally leaving for college. 

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