Gossypol, the polyphenolic constituent isolated from cottonseeds, has been used as a male antifertility drug for a long time, and has been demonstrated to exhibit excellent anti-tumor activity towards multiple cancer types. The toxic effects of gossypol limit its clinical utilization, and enzyme inhibition is an important facet of this. In the present study, in vitro human liver microsomal incubation system supplemented with UDPGA was used to investigate the inhibition of gossypol towards UGT1A1, 1A9 and 2B7-mediated metabolism of xenobiotics and endogenous substances. Estradiol, the probe substrate of UGT1A1, was selected as representative endogenous substance. Propofol (a probe substrate of UGT1A9) and 3'-azido-3'-deoxythimidine (AZT, a probe substrate of UGT2B7) were employed as representative xenobiotics. The results showed that gossypol noncompetitively inhibits UGT-mediated estradiol-3-glucuronidation and propofol O-glucuronidation, and the inhibition kinetic parameters (K(i)) were calculated to be 34.2 and 16.4 μM, respectively. Gossypol was demonstrated to exhibit competitive inhibition towards UGT-mediated AZT glucuronidation, and the inhibition kinetic parameter (K(i)) was determined to be 14.0 μM. All these results indicated that gossypol might induce metabolic disorders of endogenous substances and alteration of metabolic behaviour of co-administered xenobiotics through inhibition of UGTs' activity.