Objective: To explore the regulation function of transforming growth factor β (TGF-β1)-induced cardiac fibroblast differentiation by DNA methylation. Methods: Cardiac fibroblasts isolated from neonatal Sprague-Dawley rats were cultured and characterized using immunocytochemistry. First-passage cardiac fibroblasts were used throughout the experiment and stimulated with TGF-β1, DNA methyltransferases (DNMT), 5-aza-2′-deoxycytidine (5-aza-dC) and TGF-β-neutralizing antibody, respectively. The protein level of α-smooth muscle actin (α-SMA) was determined by Western blot assay and immunofluorescence method. The mRNA levels of α-SMA,DNMT1,DNMT3a,and DNMT3b were determined by quantitative polymerase chain reaction,and the global DNMT activity was measured. Results: TGF-β1 and 5-aza-dC both significantly upregulated the expression of α-SMA in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly down-regulated and the global DNMT activity was inhibited when treated with TGF-β1. Conclusion: Cardiac fibroblasts differentiation may be associated with the DNA methylation. Our research provides new insights in cardiac fibrosis from the perspective of epigenetics.