Microbial transformation of diosgenin (1) using Absidia coerulea yielded five new polar metabolites, which were identified as (25R)-spirost-5-en-3 beta,7 beta,12 beta,25 alpha-tetrol (2), (25S)-spirost-5-en-3 beta,7 alpha,12 beta,25 beta-tetrol (3), (25S)-spirost-5-en-3 beta,7 beta,12 beta,25 beta-tetrol (4), (25R)-spirost-5-en-3 beta,7 alpha,12 beta,25 alpha-tetrol (5), and (25R)-spirost-5-en-3 beta,7 beta,12 beta,24 beta-tetrol (6). Their structures were established on the basis of mass spectrometry and multi-dimensional NMR spectroscopy. The characteristic transformations observed were C-7 alpha, C-7 beta, C-12 beta, C-24 beta, C-25 alpha, and C-25 beta hydroxylation. The cytotoxicity of compounds 1-6 was evaluated against the human myelogenous leukemia K562 cell line and squamous cell carcinoma KB parental cell lines. Compounds 2-6 exhibited weak cytotoxicity against K562 and KB cells and were less potent than the parent compound 1.