Sai, a Konohagakure shinobi, began his life within the covert Root organization under Danzō Shimura. Orphaned young, Root subjected him to rigorous conditioning designed to erase emotions, personal connections, and individual identity; the name "Sai" itself was a temporary assignment for a mission. Trained as an efficient operative for assassination and high-risk tasks, his assignments included eliminating targets like Unmo Samidare. Despite Root's suppression, Sai maintained an interest in art, nurtured by an older Root member, Shin, whom he regarded as an adoptive brother. Their significant bond became a liability under Root regulations, forcing them toward a fatal duel to eradicate remaining emotions. Shin succumbed to illness before the fight, instructing Sai to report his death as a battle outcome and to continue suppressing feelings. Sai started a picture book depicting their experiences, placing their portraits at opposite ends with a central image of them holding hands—a detail he later forgot.

Root's conditioning left Sai outwardly emotionless, often masking his state with a false smile. He struggled profoundly with social interactions, unable to distinguish rudeness from honesty; he called Sakura "Ugly" believing nicknames fostered friendship and nearly provoked Chōji by almost using "Fat" before intervention. After joining Team Kakashi as Sasuke Uchiha's replacement, Danzō tasked Sai with infiltrating Orochimaru's ranks while ostensibly aiding in locating Sasuke. His initial interactions with Naruto Uzumaki and Sakura Haruno were confrontational, especially when questioning Naruto's loyalty to Sasuke. Sai abandoned Team Kakashi during this mission to ally with Orochimaru and deliver Danzō's proposition. However, exposure to Team Kakashi's dynamics, particularly Naruto's unwavering bonds, challenged Root's teachings. Sai began recalling suppressed memories of Shin, leading to his first genuine smile and a desire for authentic connections.

Sai's emotional development deepened, culminating in genuine rage when Shin was reanimated and weaponized by Deidara; Sai attacked the perpetrators mercilessly and shed tears upon Shin's final release. He grew observant and perceptive, recognizing Naruto's inner turmoil and Yamato's deceptions. His loyalty shifted decisively toward protecting comrades, notably supporting Naruto during the Fourth Shinobi World War. Post-war, Sai rejected Root's ideology, concluding Danzō's methods were misguided. He married Ino Yamanaka, fathered a son named Inojin, and became a caring parent, training Inojin in artistic ninjutsu while employing indirect teaching methods to foster independent growth.

In "The Lost Tower," Sai participated in a mission to capture the rogue ninja Mukade at the ancient ruins of Loran. Armed with chakra knives, he and Team Kakashi confronted Mukade, who unleashed the Ryūmyaku's chakra. As the resulting energy surge engulfed Naruto and Yamato, Sai and Sakura escaped on an ink bird. He later reunited with them after their time travel and Mukade's defeat.

Sai possesses short black hair, dark eyes, and notably pale skin. As a Root operative, he wore a black-and-gray jacket with red straps, a high-collared midriff shirt, fingerless gloves, and carried a tip-less tantō. Post-Root, his casual attire featured long-sleeved, high-collared shirts and pants. His primary abilities involve ink-based techniques, utilizing scrolls, brushes, and ninja ink to create animated creatures for combat, reconnaissance, and transportation, such as the ink bird used during the Loran mission. He also employs clones, mist techniques, and the Sealing Technique: Tiger Vision Staring Bullet—a dangerous fuinjutsu requiring emotional energy. This technique conflicted with his Root training and remained incomplete due to negative emotions.