To investigate the biotransformation of pantoprazole, a proton-pump inhibitor, by filamentous fungus and further to compare the similarities between microbial transformation and mammalian metabolism of pantoprazole, four strains of Cunninghamella (C. blakesleeana AS 3.153, C. echinulata AS 3.2004, C. elegans AS 3.156, and AS 3.2028) were screened for the ability to catalyze the biotransformation of pantoprazole. Pantoprazole was partially metabolized by four strains of Cunninghamella, and C. blakesleeana AS 3.153 was selected for further investigation. Three metabolites produced by C. blakesleeana AS 3.153 were isolated using semi-preparative HPLC, and their structures were identified by a combination analysis of LC/MS(n) and NMR spectra. Two further metabolites were confirmed with the aid of synthetic reference compounds. The structure of a glucoside was tentatively assigned by its chromatographic behavior and mass spectroscopic data. These six metabolites were separated and quantitatively assayed by liquid chromatography-ion trap mass spectrometry. After 96h of incubation with C. blakesleeana AS 3.153, approximately 92.5% of pantoprazole was metabolized to six metabolites: pantoprazole sulfone (M1, 1.7%), pantoprazole thioether (M2, 12.4%), 6-hydroxy-pantoprazole thioether (M3, 1.3%), 4'-O-demethyl-pantoprazole thioether (M4, 48.1%), pantoprazole thioether-1-N-beta-glucoside (M5, 20.6%), and a glucoside conjugate of pantoprazole thioether (M6, 8.4%). Among them, M5 and M6 are novel metabolites. Four phase I metabolites of pantoprazole produced by C. blakesleeana were essentially similar to those obtained in mammals. C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of pantoprazole.