Hydrolysis of stevioside and rebaudioside A in the gastrointestinal tract after oral intake leads to the formation of steviol, the aglycone, which is absorbed into the circulation. Although in vivo studies have shown that steviol is cleared from the body via glucuronidation, the role of liver vs. intestine in steviol glucuronidation has not been well defined and related UDP-glucuronosyltransferases (UGTs) have not been identified. The present study investigated steviol glucuronidation and obtained kinetic parameters in liver and intestinal microsomes of human and rat, as well as in recombinant human UGT systems. Results suggest that organ specificity exists in the intrinsic clearance of the glucuronidation reaction. Steviol glucuronidation was primarily mediated by UGT2B7 at low concentration and UGT2B7 and UGT1A3 at high concentration. Inhibition studies with selected UGT2B7 substrates indicate that diclofenac displayed a relatively strong inhibition (Ki, 4.2 μM) against steviol glucuronidation in human liver microsomes. Taken together, the identification of the involvement of UGT2B7 in steviol glucuronidation would provide a mechanistic basis for the evaluation of the interaction between steviol and diclofenac. As metabolic clearance of botanical-derived products can be the objects (victims) of botanical-drug interactions, further studies are needed to investigate the in vivo relevance of such interactions.