The paper is aimed to study the metabolic characteristics of osthol (Ost) in isolated hepatocytes of rat to identify which isoforms of CYP450 were responsible for Ost metabolism in vitro.  The concentration of Ost in isolated hepatocytes incubation system was determined by HPLC-UV.  The effects of incubation time, substrate concentration and hepatocytes amount on the metabolic characteristics of Ost were investigated.  CYP2C8 inhibitor quercetin (Que), CYP2C9 inhibitor sulfaphenazole (Sul), CYP2D6 inhibitor yohimbine (Yoh), CYP3A4 inhibitor troleandomycin (Tro) and CYP450 inducer rifampicin (Rif) were used to investigate their    effects on the metabolism of Ost.  The metabolism of Ost in isolated rat hepatocytes showed an enzymatic    kinetic characteristics.  Rif induced Ost elimination in rat hepatocytes; Yoh, Sul, Que did not have effects on Ost metabolism in vitro.  Between 0−200 μmol·L⁻¹, Tro inhibited Ost metabolism in a concentration-dependent manner.  CYP3A4 is the enzyme metabolizing Ost in vitro; CYP2C8, CYP2C9 and CYP2D6 did not involve in Ost metabolism in rat hepatocytes.