N-Glucosidation is known as a major metabolic reaction for barbiturates in humans. However, the enzyme(s) involved in this N-glucosidation has not been clarified yet. Thus, to clarify the enzyme(s) involved in the N-glucosidation in human liver microsomes, we investigated the N-glucosyltransferase activity in recombinant UDP-glucuronosyltransferases (UGTs) using AS-3201, an aldose reductase inhibitor, as a substrate. AS-3201 was found to be biotransformed to both N-glucoside and N-glucuronide in human liver microsomes. The N-glucosyltransferase activities were detectable with multiple UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT2B4, UGT2B7, and UGT2B15). In contrast, the N-glucuronyltransferase activities for the same substrate were seen with UGT1A (UGT1A1, UGT1A3, UGT1A4, and UGT1A9) but not UGT2B isoforms. We then determined the relative activity factor of each recombinant UGT and estimated the contribution of each UGT isoform to the N-glucosidation in human liver microsomes. The results showed that UGT2B isoforms mainly contribute to AS-3201 N-glucosidation in human liver microsomes. In addition, the activity of AS-3201 N-glucosyltransferase significantly correlated with that of amobarbital N-glucosyltransferase in microsomes from sixteen human livers (r=0.964, P<0.01), indicating that UGT2B isoforms were also involved in the barbiturate N-glucosidation in humans. The findings of this study clearly show that UGT2B specifically utilizes UDP-glucose but not UDP-glucuronic acid as a sugar donor for the conjugation of AS-3201 in human liver microsomes.