PURPOSE: To prepare an androstane compound by culturing a mutant of Mycobacterium vaccae in a medium containing a sterol as a substrate.

CONSTITUTION: A mutant of Mycobacterium vaccae, wherein the androstane compound-decomposition enzyme existing in the wild strain is absent or present only as a deactivated state, such as Mycobacterium vaccae MCI-1102 (FERM-P No. 4739) or MCI-1103 (FERM-P NO.4740), is inoculated in a medium containing sterol, its C-3 ester derivative, C-3 ether derivative or their oxidation intermediates as a substrate, and cultured to accumulate an androstane compound such as androsta- 1,4-diene-3,17-dione, androst-4-ene-3,7-dione, etc. in the medium.