The present study has investigated the effect of tacrolimus on the pharmacokinetics of an active metabolite of irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38) and SN-38 glucuronide (SN-38G) in rats. The effect of tacrolimus on SN-38 glucuronidation was also investigated in human and rat liver microsomes. When tacrolimus (0.5 mg/kg) was intravenously injected in rats 15 min before intravenous injection of CPT-11 (5 mg/kg), tacrolimus decreased the plasma concentration of SN-38G. Tacrolimus significantly decreased the area under plasma concentration-time curve (AUC) of SN-38G without change in the mean residence time. On the contrary, significant changes in the pharmacokinetic parameters of SN-38 were not observed. SN-38 glucuronidation in human and rat liver microsomes was inhibited dose-dependently by the presence of tacrolimus and the 50% inhibition concentration (IC₅₀) values of tacrolimus in rat and human liver microsomes were 10.33 μM and 3.58 μM, respectively. When the inhibition type was determined by Lineweaver-Burk and Dixon plots, the inhibition was noncompetitive and the calculated inhibition constant (K_i) values for rat and human liver microsomes were 12.57 μM and 3.88 μM, respectively. These findings suggest that tacrolimus inhibits UGT1A1-mediated SN-38 glucuronidation. Considering the IC₅₀ and K_i values for tacrolimus, it is likely that tacrolimus does not alter the pharmacokinetics of SN-38 and SN-38G at the clinically used dosages, suggesting the possibility that tacrolimus can use safely for cancer patients with irinotecan chemotherapy.