PURPOSE: To obtain the titled compound useful as an antitumor agent, etc., on an industrial scale at a low cost, by culturing a microbial strain belonging to Streptomyces genus, having mitomycin B productivity and deficient in the productivity of mitomycin A and C and collecting the produced objective substance from the cultured product.

CONSTITUTION: A microbial strain belonging to Streptomyces genus, having mitomycin B productivity and deficient in the productivity of mitomycin A and C [e.g. Streptomyces caespitosus ATCC 27422 (FERM BP-1290)] is cultured in a medium under aerobic condition at 30°C under shaking to produce and accumulate mitomycin B in the cultured product. The accumulated mitomycin B is separated from the cultured product to obtain mitomycin B by fermentation process.