Aim To investigate the effect of metformin on angiotensin Ⅱ （AngⅡ） -induced proliferation of adult rat cardiac fibroblasts （CF） and its underlying mechanism. Methods The adult rat CF was isolated by a combination of trypsin and collagenase Ⅱ digestion.The cell proliferation was induced by AngⅡ （100 nmol/L） stimulation,and the CF was treated with metformin in different concentrations （10,50,and 200 μmol/L）.The proliferation of CF was evaluated by MTT assay,and the DNA synthesis was detected by EdU incorporation.The expression of endothelial nitric oxide synthase （eNOS） and phosphorylated endothelial nitric oxide synthase （p-eNOS） were detected by Western blot.The level of NO in CF culture supernatant fluids was measured by nitrate reductase method. Results Stimulation with AngⅡ for 48 h induced the proliferation of adult rat CF,and this effect was inhibited by pretreatment of CF with metformin in a concentration-dependent manner.Metformin significantly increased the phosphorylation level of eNOS in CF as well as the level of NO in cell culture supernatant fluids in a concentration-dependent manner.In addition,pretreatment of CF with eNOS inhibitor L-NAME markedly attenuated the inhibitory effect of metformin on AngⅡ-induced cell proliferation. Conclusion Metformin inhibits AngⅡ-induced proliferation of adult rat CF,and this effect may be associated with the activation of the eNOS/NO pathway.