Isaburo Sasaki is the commander of the Mimawarigumi, an elite police force in the service of the Bakufu. Born as the main heir into the prestigious Sasaki family, he is considered a pure product of the upper class and a well-rounded elite, often referred to as the Monster of Three Heavens for his mastery of the sword, the gun, and the pen. He is also the older half-brother of Sasaki Tetsunosuke, whom he treats with open contempt as the family's failure due to his status as a child from a concubine.

On the surface, Isaburo presents a calm, serious, and somewhat arrogant demeanor. He speaks in a polite but condescending tone and considers himself part of a superior elite class, regularly looking down on those he deems unworthy. He often has half-opened eyes and wears a monocle over his right eye, which contributes to his elusive and impassive expression. However, this cold exterior masks a deeply wounded and cynical individual. A notable quirk of his personality is an extreme addiction to mobile phones and email. He will forcibly register his email address on the phones of others under the name Sabu-chan, claiming he does not have many friends. His emails are often filled with awkwardly cheerful phrases and emoticons, creating a humorous gap between his serious public image and his lonely, almost desperate private attempts at connection.

The driving force behind Isaburo's actions is a profound personal tragedy. In the past, he was involved with Matsudaira in the founding of the Roshigumi, which would later become the Shinsengumi. While working alongside the Naraku, the shogunate's secret assassin organization, he was part of a plot to have the Hitotsubashi family assassinated. However, moved by the genuine spirit of Kondo Isao and his men, Isaburo chose to betray the plan and sabotage the assassination. As punishment for his disobedience, Oboro of the Naraku sent an assassin to kill Isaburo's pregnant wife and unborn daughter. The young assassin sent was Mukuro, a child who would later become Imai Nobume. This event broke Isaburo. He developed a hatred for the corrupt system that allowed such a thing to happen and resolved to destroy it from within. To that end, he kept Nobume, the instrument of his family's death, as his assistant, giving her the name he had intended for his own daughter. He then allied himself with Takasugi Shinsuke and the Hitotsubashi faction, pretending to serve the shogunate while secretly planning to plunge the country into chaos and revolution.

Isaburo's primary role in the story is as a major antagonist, first appearing prominently during the Baragaki arc. In this arc, he manipulates his half-brother Tetsunosuke and the Shinsengumi to root out a terrorist group, all while using the situation to test and provoke Hijikata Toushirou. He later becomes Director of Police after the ascension of Hitotsubashi Nobu Nobu and orchestrates a larger conflict to force a final confrontation between his forces, the Shinsengumi, and the Naraku. His key relationships are defined by his trauma. He shares a complex bond with Imai Nobume, whom he keeps as a tool but comes to genuinely care for as a surrogate daughter, forbidding her from smiling until her debt of killing his family is repaid. He holds a secret envy and admiration for the Shinsengumi, particularly Kondo Isao, viewing their unpolished but true samurai spirit as something genuine that his own elite status could never provide. His connection to Gintoki begins as a frustrating email correspondence, but he eventually respects the Yorozuya's strength and convictions.

Throughout his story, Isaburo undergoes a subtle but powerful development. He begins as a man consumed by a desire for revenge and self-destruction, seemingly willing to sacrifice anything, including his own half-brother, to tear down the current system. However, his interactions with the Shinsengumi and his growing attachment to Nobume chip away at his nihilism. In his final battle, he chooses to protect Nobume and his brother Tetsunosuke, making a sacrifice to ensure their escape. He dies with a satisfied expression, recognizing that his wish for the future, embedded in the name Nobume, had finally been fulfilled. His abilities are formidable, as he is a triple threat capable of overwhelming even a skilled swordsman like Hijikata Toushirou in combat. His legacy is a complex one of a man who embraced darkness in a desperate attempt to create a better world, earning a degree of respect from both his enemies and allies after his death.