Two years after moving to San Francisco, Riley is now 13 and preparing to start high school. Her emotions—Joy, Sadness, Fear, Anger, and Disgust—have developed a new part of her mind called the "Sense of Self," which contains the memories and feelings that define her core personality. Joy has created a device to send negative memories to the back of Riley's mind. Riley, along with her best friends Bree and Grace, is invited to a weekend hockey camp, hoping to make her school’s team, the Firehawks. The night before the camp, a "Puberty Alarm" sounds, and mind workers arrive to upgrade the emotion console.

The emotions soon realize that Riley is overreacting to all their inputs. Four new emotions—Envy, Embarrassment, Ennui, and Anxiety—arrive and introduce themselves. Initially friendly, the old and new emotions clash, especially as Joy believes Riley should enjoy the camp, while Anxiety wants her to secure a spot on the team and make new friends, especially since Bree and Grace will be attending a different high school.

Feeling Riley needs a change, Anxiety moves the Sense of Self to the back of her mind and locks up the old emotions in a "memory vault." The new emotions then create a corrupted Sense of Self using negative memories and encourage Riley to befriend a popular hockey player, Val, by mimicking her, straining her relationships with Bree and Grace. The old emotions escape, and Sadness returns to Headquarters while the others retrieve the original Sense of Self.

Sadness reaches Headquarters but cannot stop Riley from sneaking into her coach’s office and reading her notebook, where she learns the coach doesn't think she's ready to join the Firehawks. Anxiety, seeking more control, pushes Riley to the edge. The emotions retrieve the original Sense of Self from a mountain of bad memories. To get back to Headquarters, they cause an avalanche, riding it back, but the memories spill and further corrupt Riley's Sense of Self. Anxiety's actions lead Riley to a state of self-doubt, causing her to panic during a crucial hockey game, where she hogs the puck, misses shots, and accidentally hurts Grace, landing her in the penalty box. Overwhelmed, Riley has a panic attack.

The old emotions return to Headquarters, and Joy persuades Anxiety that Riley doesn’t need to change to have a better future. Anxiety steps back, allowing the original Sense of Self to be reinstalled, but Riley’s attack continues. Joy realizes that no single emotion can define Riley and removes the first Sense of Self, allowing a new, more complex sense to form from all her memories. The emotions embrace this third Sense of Self, stabilizing it, and Riley calms down, reconciling with Bree and Grace. Riley, now in control of her emotions, asks Joy to take charge and finishes the game with a smile.

Later, Riley starts high school, befriends Val and the Firehawks, and maintains her friendships with Bree and Grace. The old and new emotions work together to protect Riley's evolving Sense of Self. Riley reads an email about making the Firehawks and looks at herself in the mirror with pride and happiness.