Hydroxylation of steroids at various positions is a powerful tool for the production of valuable pharmaceutical ingredients and precursors. Our paper reported the synchronous dihydroxylation of an efficient strain, i.e., Colletotrichum lini AS3.4486, at two points. C. lini AS3.4486 was selected from 10 strains; this strain can catalyze the dihydroxylation of androst-1,4-dien-3,17-dione at C-11 alpha and C-15 alpha positions. Transformation of ADD(I) by C lini AS3.4486 produced metabolites II-IV. The structures of these compounds were elucidated by liquid chromatography mass spectrometry (LC-MS), Fourier Transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and X-ray as 15-hydroxyandrost-1,4-dien-3,17-dione (15 alpha-OH-ADD; II), 11,15-dihydroxyandrost-1,4-dien-3,17-dione (11,15-diOH-ADD; III), and 15,17 beta-dihy-droxyandrost-1,4-dien-3-one (15-OH-BD; BD is the abbreviation of boldenone; IV). III, as a novel compound, was reported for the first time. The course of conversion and mechanism about dihydroxylation reaction was also investigated. On the basis of time course analysis of hydroxylation, I underwent regioselective hydroxylation at 15 position and was subsequently converted to III and IV. Enzyme inhibition analysis showed that 11- and 15-hydroxylation were catalyzed by different hydroxylases. The effect of substrate concentration on I transformation was also determined. Results showed that the optimum concentration of I was 20 and the yield of III was up to 18.8 g/L. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.