Objective To investigate the metabolites of scopoletin using a rat liver microsomal incubation system and the effects of drug-containing serum on rat hepatic stellate cells (HSC). Methods The metabolites of scopoletin from the rat liver microsomal incubation system were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and the effects of drug-containing serum on rat HSC were observed. The metabolites of scopoletin were analyzed by high-resolution second-order mass spectrometry with an ACQUITY UPLC HSS T3 column (100 mm×2. 1 mm i. d.d., 1. 8 μm) and 0. 1% formic acid aqueous solution-0. 1% formic acid acetonitrile mobile phase. Annexin V-APC single staining and MTT method were used to observe the induction of apoptosis and inhibition of proliferation of HSC induced by drug-containing serum. Results Three metabolites of scopolactone were identified with the rat liver microsome incubation system, which indicated that the main metabolic pathway was demethylation and glucuronidation. The drug-containing serum had a weak effect on inducing HSC apoptosis and inhibiting HSC proliferation. Conclusions Combined with previous studies, the anti-fibrotic effects of scopolactone do not directly affect HSC, and further study is needed to clarify the possible mechanism.