The inter-species differences of thienorphine metabolism were investigated in human, Beagle  dog and rat liver microsomes, by comparing enzyme kinetics of the parent drug and the formation of its major metabolites.  The incubation systems of thienorphine with liver microsomes of the three species were optimized in terms of thienorphine concentration, microsomal protein content and incubation time.  The concentrations of thienorphine and its metabolites in incubates were measured by a LC-MS/MS method.  The biotransformation of thienorphine by human liver microsomes was the lowest among the three species.  The K_m, V_max, CL_int and T_1/2 of thienorphine obtained from human liver microsomes were (4.00 ± 0.59) µmol·L⁻¹, (0.21 ± 0.06) µmol·L⁻¹·min⁻¹, (117 ± 3.19) mL·min⁻¹·kg⁻¹ and (223 ± 6.10) min, respectively.  The corresponding kinetic  parameters for dog and rat liver microsomes were (3.57 ± 0.69) and (3.28 ± 0.50) µmol·L⁻¹, (0.18 ± 0.04) and (0.14 ± 0.04) µmol·L⁻¹·min⁻¹, (213 ± 1.06) and (527 ± 7.79) mL·min⁻¹·kg⁻¹, (244 ± 1.21) and (70.7 ± 1.05) min, respectively.  A total of six phase I metabolites were observed in liver microsomes, including one N-dealkylated metabolite, three oxidative metabolites and two N-dealkylated oxidation metabolites.  All these six metabolites were detected in the liver microsomes of the three species.  However, the relative amounts of the metabolites generated were different in three species.  The results indicated that the major phase I metabolic pathway of thienorphine was similar in the liver microsomes from all three species.  However, the inter-species differences observed were relative amounts of the metabolites as well as the metabolic characteristics of thienorphine in liver microsomal incubates.