OBJECTIVE： To study in vitro metabolism pathway of effective component of Bletilla striata as Militarine in liver microsomes and kinetics characteristics of enzyme-catalyzed reactions. METHODS： The in vitro incubation system of rat and human liver microsomes was established， and incubation reaction of Militarine was performed. UPLC-QTOF-MS was used to identify the structure of its metabolites in combination with UNIFI database and references. Using puerarin as internal standard， UPLC-Triple Quad-MS was used to quantitatively analyze metabolic transformation of Militarine in rat liver microsomes. The kinetic parameters （vmax， km， CLint） of Militarine enzyme-catalyzed reactions with/without reducing coenzyme Ⅱ （NADPH） were calculated by fitting the curves with GraphPad Prism 5.0 software. RESULTS： After incubation in rat and human liver microsomes， Militarine produced a chemical formula C21H29O11， which was presumed to be a metabolite of Militarine ester bond hydrolysis. The kinetic study of enzyme-catalyzed reactions showed that vmax of Militarine enzyme-catalyzed reactions with/without NADPH were 1.955， 2.129 nmol/（h·mg）； km were 8.601， 9.854 nmol/mL； CLint were 0.227 3， 0.216 1 mL/（h·mg）； there was no significant difference between with NADPH and without NADPH. CONCLUSIONS： The main metabolic pathway of Militarine in liver microsomes is the hydrolysis of C1 and C4 ester bonds. Its metabolism does not depend on the pathway of cytochrome P450 enzymes initiated by NADPH.