Misao Makimachi is a teenage kunoichi, or female ninja, and the youngest member of the Oniwabanshū, a clan of onmitsu based in Kyoto. She is sixteen years old when she first appears, though she looks younger. She is the granddaughter of the former Okashira, or leader, of the Oniwabanshū. From the age of eight, she was raised by Shinomori Aoshi until he and several other members left her in the care of Kashiwazaki Nenji, known as Okina, the elder guardian of the clan. She shares a strong bond with Okina but continues to view Aoshi as the best. She also holds a special affection for Han'nya, who taught her how to fight.

Misao is bright, energetic, and often impulsive. She is frequently compared to a weasel due to her quick movements and spirited nature. She can be headstrong and speaks her mind without hesitation. Beneath her lively exterior, she is deeply loyal, compassionate, and fiercely protective of her friends and her clan. Her primary motivation is her admiration and romantic feelings for Shinomori Aoshi. She leaves Kyoto to search for him after learning he is in Tokyo and instead encounters Himura Kenshin during her journey. She travels with Kenshin back to Kyoto, hoping that he will lead her to Aoshi.

In the story, Misao serves as a key ally during the Kyoto arc, which takes a darker turn in the narrative. She becomes a close friend to Kenshin, Kamiya Kaoru, and Myōjin Yahiko, helping them fight the agents of Makoto Shishio. After learning that Aoshi attempted to kill Okina, Misao struggles to accept the change in the man she once revered. She declares herself the new Okashira of the Oniwabanshū, refusing to recognize Aoshi as the clan's leader any longer. Kenshin promises to bring Aoshi back to her, a vow that moves her deeply. She later proves herself a capable and determined leader.

Misao's key relationships are defined by loyalty. She admires Aoshi above all others, though their bond becomes strained by his actions. She shares a fatherly connection with Okina, who remains a steadfast guide. She becomes a close companion to Kenshin, seeing him as a mentor of sorts, and forms a strong friendship with Kaoru and Yahiko. Her relationship with Han'nya is especially formative, as he was her caretaker and combat instructor.

Over the course of the story, Misao matures from an impulsive girl into a more thoughtful and strategic fighter. While still retaining her bright spirit, she learns to temper her emotions and take on greater responsibility, including stepping into the role of leader for the Oniwabanshū when the clan is in need.

As a kunoichi, Misao is trained in stealth, agility, and hand-to-hand combat. Her primary weapon is the kunai, and she also uses shuriken. She is a skilled fighter for her age, capable of defeating multiple opponents and contributing effectively in battle alongside more experienced warriors. Her movements are quick and nimble, reflecting her ninja training, and she uses her small stature to her advantage in combat.