Abstract: Objective To investigate the effect of senkyunolide I on cholestatic liver injury through the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods A total of 30 mice were divided into three groups: a normal group, a cholestatic liver injury model group, and a senkyunolide I treatment group. The model group was fed a diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), while the senkyunolide I group was additionally administered with 100 mg/kg of senkyunolide I by gavage daily. The mice were treated four weeks. Then, blood samples were collected, and the levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. The mice were sacrificed and liver tissues were collected for hematoxylin-eosin (H-E) staining. The levels of related proteins and the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and superoxide dismutase (SOD) were detected by Western blot and RT-qPCR. Furthermore, HepG2 cells were divided into three groups: a normal group, a model group, and a senkyunolide I group. The senkyunolide I group was treated with 100 μmol/L senkyunolide I for 24 h for apoptosis assay. The levels of janus kinase 2 (JAK2) and STAT3, and their phosphorylated proteins and mRNA were measured. Results Compared with the normal group, the model group exhibited significantly elevated serum AST and ALT levels(P＜0.01). Compared with the model group, the senkyunolide I group showed significantly reduced AST and ALT levels(P＜0.01). Compared with the normal group, the model group presented decreases in liver SOD and catalase (CAT) activity, and increases in malondialdehyde (MDA) levels(P＜0.01). Compared with the model group, the senkyunolide I group showed increases in SOD and CAT activity and decreases in MDA levels(P＜0.01). Compared with the normal group, phosphorylated STAT3 levels were elevated in the model group(P＜0.05). Compared with the model group, phosphorylated STAT3 levels were reduced in the senkyunolide I group(P＜0.05). According to cellular experiment results, hepatocyte apoptosis increased in the model group compared with those in the normal group. Compared with the model group, the senkyunolide I group showed decreased cellular apoptotic rates, enhanced SOD and glutathione (GSH) activity, lowered MDA levels(P＜0.01), and upregulated mRNA expression of Nrf2, HO-1, and SOD(P＜0.05), with enhanced antioxidant capacity, reduced JAK2 and STAT3 phosphorylation(P＜0.05), and alleviated hepatocyte damage. Conclusions Senkyunolide I alleviates cholestatic liver injury through regulating the STAT3 signaling pathway, inhibiting oxidative stress, enhancing antioxidant enzyme activity, and reducing hepatocyte apoptosis.