Microbial transformation of diosgenin((25R)-spirost-5-en-3β-ol)using Cunninghamella blakesleana AS 3.970,afforded eleven polyhydroxylated derivatives,including seven previously unreported steroids,such as 25(R)-spirost-5-en-3β,7α,12β-triol(1),25(R)-spirost-5-en-3β,7α,12β,15α,21-pentaol(3),25(R)-spirost-5-en-3β,7α,12β,18-tetraol(4),25(R)-spirost-5-en-3β,7α,9α,15α-tetraol(5),25(R)-spirost-5-en-3β,7α,11β,21-tetraol(6),25(R)-spirost-5-en-3β,7β,15α,21-tetraol(8),and 25(R)-spirost-5-en-3β,7β,12β,18-tetraol(10).The structures of metabolites 1?11 were elucidated by 1D-,2D-NMR as well as HR-ESIMS techniques.Additionally,the biotransformation time-course of diosgenin by C.blakesleana AS 3.970 was also investigated.And the transformation pathway was also proposed on the basis of structural analyses and biotransformation time-courses.The P-glycoprotein(P-gp)inhibitory effects of these metabolites 1?11 were evaluated in adriamycin resistant human breast adenocarcinoma cell line(MCF-7/ADR)at 20 μmol·L-1.And compounds 4 and 6 could increase the accumulation of adriamycin in MCF-7/ADR cells approximately four times of control group with the better water-solubility,which suggested the significant potential P-glycoprotein inhibitory activities of 4 and 6.In silico docking analysis suggested that compound 4 had similar P-gp recognition mechanism with verapamil(a classical inhibitor).