Timosaponin BII (TBII) is an active constituent of some Chinese medicinal herbs, and is abundant in Anemarrhena asphodeloides Bunge. To better understand the pharmaceutical mechanisms of TBII, we prepared its deglycosylated derivatives through biotransformation with fungi Colletotrichum gloeosporioides, Acremonium alternatum and Aspergillus niger. In the presence of 10 g/L glucose, transformation with C. gloeosporioides yielded 4 products: timosaponin AIII (P3) and AI (P5) and their beta isoforms (P2 and P4 respectively). However, P4 and P5 could not be detected when 0-5 g/L glucose was applied; P2 and P3 disappeared at 20 g/L glucose. P4 is a newly discovered compound. We firstly report that fungi in whole cell model can hydrolyze C3-Omicron-glucopyranosyl residues of steroidal saponins after removing the C26 sugar. Therefore, the transformation efficiency depends on glucose. Transformation of TBII, and maybe other steroidal saponins, with C. gloeosporioides is an ideal method to prepare the metabolites in vitro. (C) 2015 Elsevier Ltd. All rights reserved.