Kitasato began his study of medicine at Igakusho Hospital (now Kumamoto Medical School). When his mentor, Dutch physician C.G. van Mansvelt, left the school, Kitasato entered Tokyo Medical School (now the Faculty of Medicine, University of Tokyo). After graduation (M.D., 1883) he carried out bacteriological research at the Central Sanitary Bureau of the Ministry of Home Affairs.

In 1885 Kitasato moved to Berlin to join the laboratory of German bacteriologist Robert Koch (Koch, Robert). There, with Emil von Behring (Behring, Emil von), he studied tetanus and diphtheria, two bacterial infections that cause symptoms through the secretion of toxins. In 1889 Kitasato succeeded in obtaining the first pure culture of the tetanus bacteria (bacilli), and the following year he and von Behring demonstrated that immunity to tetanus could be achieved by injecting a susceptible animal with serum containing antitoxin produced in the blood of an animal exposed to the bacterial toxin. They soon successfully applied this approach, called serum therapy, to the treatment of diphtheria.

Returning to Japan in 1892, Kitasato founded and became president of the Institute for Infectious Diseases, a laboratory near Tokyo that was incorporated in 1899 into the Ministry of Home Affairs. The next year he founded Yojoen, a sanatorium for victims of tuberculosis, and concurrently served as president of both organizations.

Kitasato was sent to Hong Kong in 1894 to investigate an outbreak of the bubonic plague. Within a month he identified the causative organism of the plague, the bacillus Pasteurella pestis (now called Yersinia pestis; renamed after French bacteriologist Alexandre Yersin, who independently discovered the plague bacillus during the Hong Kong epidemic).

In 1914 Kitasato resigned the directorship of the imperial institute and founded the Kitasato Institute. He became the first dean of the medical school of Keio University, an institution he helped establish, in 1917 and held this position until 1928. When the Japanese Medical Association was founded in 1923, he became its first president. In 1924 the emperor invested him with the title of baron.