Abstract: AIM　To investigate the hydroxylation process of benproperine in humans. METHODS After an oral administration of 60 mg benproperine to ten healthy male volunteers, urine samples collected within 0～24 h were extracted by solid phase extraction and analyzed by liquid chromatography-ion trap mass spectrometry. A microbial transformation of benproperine combined with semi-preparative HPLC was used to get two reference substances of the hydroxylated metabolites, and their structures were then elucidated by NMR. Furthermore, the structures of conjugated metabolites were speculated based on their characteristics in MS fragmentation. RESULTS　Five hydroxylated metabolites of benproperine and some of their conjugates with endogenous glucuronic acid or sulfuric acid were found in urine of volunteers after the dose. The structures of two metabolites were identified as 4″-hydroxybenproperine and 4-hydroxybenproperine by comparison of HPLC retention times and mass spectra with those of authentics obtained from the microbial transformation. CONCLUSION　Hydroxylation of benproperine occurs preferentially at para-position of the alkoxyl group in the aromatic ring. The hydroxylated metabolites of benproperine in human urine mainly exist as their glucuronic acid or sulfuric acid conjugates.