Objective To reveal the expression pattern of miR-325-3p in hepatopulmonary syndrome (HPS) and analyze the mechanism of its effect on pulmonary angiogenesis. Methods The experimental animals were specific pathogen-free (SPF)-grade male Sprague-Dawley (SD) rats (8 weeks old, weighing 300-350 g). The rat model of HPS was established by chronic common bile duct ligation. A total of 57 HPS rats were randomly divided into HPS group (n=12), HPS+NC-agomir group (n=12), HPS+miR-325-3p-agomir group (n=11), HPS+NC-antagomir group (n=11), and HPS+miR-325-3p-antagomir group (n=11), rats in Sham group were used as control (n=12). Rats in Sham group and HPS group were injected with 200 μL 0.9% normal saline through tail vein. Rats in HPS+NC-agomir group, HPS+miR-325-3p-agomir group, HPS+NC-antagomir group, and HPS+miR-325-3p-antagomir group were injected 200 μL NC-agomir, miR-325-3p-agomir, NC-antagomir, miR-325-3p-antagomir through tail vein, respectively. The injections were given twice a week for 4 weeks. PaO2 and AaDO2 were analyzed using a blood gas analyzer. The levels of TGF-β1, IL-6, TNF-α and NO in lung tissue were measured according to manufacturer's instructions. The lung tissue was stained with HE staining kit. miR-325-3p and mRNA levels of Serpin B1, ET-1, iNOS, VEGF and vWF and ANG-2 were detected by qRT-PCR. The protein levels of Serpin B1, ET-1, iNOS, VEGF, vWF, ANG-2, ERK1/2, p-ERK1/2, STAT3 and p-STAT3 in lung tissue were detected by Western blot. The expression of VEGF in lung tissues was detected by immunofluorescence staining. The interaction between miR-325-3p and Serpin B1, ERK1/2 and STAT3 was explored by multi-factor regression analysis. Pearson correlation analysis was used to explore the relationship between miR-325-3p and pulmonary angiogenesis.Results Compared with HPS group and HPS+NC-agomir group, the level of miR-325-3p and PaO2 in HPS+miR-325-3p-agomir group was increased (P＜0.05), the levels of AaDO2, TGF-β1, IL-6, TNF-α and NO were decreased (P＜0.05), lung tissue damage was reduced, the relative levels of Serpin B1, ET-1, iNOS, VEGF, vWF and ANG-2 mRNA and protein were decreased (P＜0.05), the relative fluorescence intensity of VEGF and the relative phosphorylation levels of ERK1/2 and STAT3 were decreased (P＜0.05). Compared with HPS group and HPS+NC-antagomir group, the level of miR-325-3p and PaO2 in HPS+miR-325-3p-antagomir group was decreased (P＜0.05), the levels of AaDO2, TGF-β1, IL-6, TNF-α and NO were increased (P＜0.05), lung tissue damage was aggravated, the relative levels of Serpin B1, ET-1, iNOS, VEGF, vWF and ANG-2 mRNA and protein were increased (P＜0.05), the relative fluorescence intensity of VEGF and the relative phosphorylation levels of ERK1/2 and STAT3 were increased (P＜0.05). Serpin B1 protein and phosphorylation level of STAT3 had statistically significant effects on miR-325-3p (P＜0.05). miR-325-3p was significantly negatively correlated with ET-1, iNOS, VEGF, vWF and ANG-2 protein (P＜0.01). Conclusion miR-325-3p targets Serpin B1 to inhibit pulmonary angiogenesis in HPS