Born in Oguni, Japan, Kitasato graduated from the medical school of the University of Tokyo in 1883 and then went to Berlin to study under Robert Koch. A close and long-lasting friendship developed between the two men.

While in Berlin Kitasato worked with Emil von Behring and in 1890 they announced the discovery of antitoxins of diphtheria and tetanus. They showed that if nonimmune animals were injected with increasing sublethal doses of tetanus toxin, the animals became resistant to the disease. Their paper laid the basis for all future treatment with antitoxins and founded a new field in science, that of serology. Kitasato returned to Japan and became director of the Institute of Infectious Diseases in 1892. Two years later there was an outbreak of bubonic plague in Hong Kong and he succeeded in isolating the plague bacillus, Pasteurella pestis. In 1898 he isolated the microorganism that causes dysentery.

He founded the Kitasato Institute for Medical Research in 1914 and became dean of the medical school, Keio University, Tokyo. In 1924 he was created a baron. In 1908 Koch visited Japan and Kitasato secretly obtained clippings of the visitor's hair and fingernails. When Koch died in May 1910, Kitasato built a small shrine for the relics in front of his laboratory; when Kitasato died, his remains were placed in the same shrine, next to those of his respected master.