Abstract: Incubation of 16α-Methyl-5α-△⁹⁽¹¹⁾-Pregnene-3β, 17α, 21-triol-20-one 3β, 21-diacetate (Ⅲ)with a mixed culture of Mycobacterium smegmatis (MS₁) and Arthrobacter simplex (A₁) gave 16α-methyl-1,4,9(11) Pregnatriene-17α, 21-dihydroxy-3,20-dione (Ⅳ) (65% yield) and a small amount of 16α-methyl-△^1,4-pregnadiene-9α, 11α-epoxy-17α, 21-diol-3, 20-dione (Ⅴ). At 0.5% substrate concentration, Ni ion was shown to be needed to inhibit the production of the byproduct compound(Ⅴ) and increase the yield of compound(Ⅳ).