Makoto Niijima is a third-year student at Shujin Academy who serves as the student council president. She is the younger sister of Sae Niijima, a prosecutor. Makoto lives with her sister after their father passed away, and the loss has shaped her sense of responsibility and her desire to live up to expectations. She is highly intelligent, honest, and hardworking, with a perfectionist streak and a strong sense of duty. She is known for being serious and principled, but she also has a hidden meek side and can be frightened by things like ghost stories and the dark. Her serious demeanor sometimes causes her to feel isolated from her peers, and she struggles with the pressure of being a model student who follows the path set by adults without question.

Makoto's initial motivation is to secure a good future for herself, which leads her to accept the principal's request to investigate the Phantom Thieves in exchange for a university recommendation. However, her deeper motivation is a desire for justice and a need to find a place where she truly belongs. She resents being used by authority figures and feels trapped by the rigid expectations placed on her. After meeting the Phantom Thieves and witnessing their fight against corruption, she becomes determined to challenge the societal injustices she sees. Her personal journey is about breaking free from the role of the obedient student and learning to act on her own convictions.

In the story of Persona 5 the Animation: Dark Sun, Makoto initially appears as an antagonist to the Phantom Thieves, tasked with exposing their identities. She discovers the identities of the protagonist, Ryuji Sakamoto, Ann Takamaki, and Yusuke Kitagawa, and uses this knowledge to blackmail them into helping her take down the mafia boss Junya Kaneshiro. During the infiltration of Kaneshiro's Palace, Makoto is trapped and blackmailed herself, but this crisis leads to her awakening her Persona, Johanna. She joins the Phantom Thieves under the code name Queen, and her analytical mind quickly establishes her as the group's strategist and advisor. Her role is to plan heists, gather intelligence, and provide tactical direction during missions.

Key relationships include her bond with her sister Sae, which is fraught with tension due to Sae's overbearing expectations and Makoto's fear of being a burden. Their relationship evolves as Makoto confronts Sae's distorted desires within her Palace. Within the Phantom Thieves, Makoto develops a close partnership with the protagonist, who becomes a trusted confidant. She also learns to open up to others, particularly Ann Takamaki, with whom she reconciles after initial friction. Her interactions with Ryuji Sakamoto highlight her strict personality clashing with his impulsiveness, though she ultimately respects his passion.

Makoto undergoes significant development throughout the story. She starts as a reserved and insecure young woman who follows orders without question, but her experiences with the Phantom Thieves give her confidence and a sense of purpose. She learns to trust her own judgment, stand up for what she believes in, and prioritize her own values over external validation. This growth culminates in her facing her sister and helping to change her heart, as well as actively participating in the final confrontation against the godlike entity controlling society.

Makoto's notable abilities include her signature Persona, Johanna, which takes the form of a motorcycle. Johanna wields nuclear-elemental skills, which are effective against enemies afflicted with burn, freeze, or shock. Makoto is also capable of using healing skills to support her party, making her a versatile combatant. Her Phantom Thief code name Queen reflects her leadership qualities, and she fights using tekko knuckle weapons and revolvers. Her strategic mind allows her to analyze enemy weaknesses and devise effective battle plans. As she grows stronger, her Persona evolves into Anat and later Agnes, enhancing her nuclear abilities and unlocking more powerful support skills.