Abstract: AIM:To investigate the role of Notch3 in the transformation of cardiac fibroblasts (CFs) to myofibroblasts (MFs). METHODS: CFs were isolated by collagenase digestion method and incubated for 24 h with different concentrations of TGF-β1. Western blot analysis was used to measure α-smooth muscle actin (α-SMA) and Notch3 expression. ELISA was used to measure hydroxyproline content in the supernatants and real-time PCR was used to measure Notch3 mRNA expression. RNAi method was employed to interfere with the expression of Notch3, and Western blot and real-time PCR analysis were employed to analyze the interference effects. RESULTS: TGF-β1 induced increases of hydroxyproline content in the supernatants and α-SMA expression dose dependently in CFs and induced decreases of Notch3 expression. Compared with those in control group, significant differences were observed in the changes at the concentrations of 2, 5, and 10 ng/ml (all P<0.05). Notch3 mRNA and protein expression decreased, and hydroxyproline content in the supernatants and α-SMA expression increased in interference group (all P<0.05). CONCLUSION: Notch3 expression downregulation can induce the transformation of CFs to MFs.