〔Abstract〕 Objective To investigate the role of N-methyladenosine(m6A) demethylase ALKBH5 in the proliferation and activation of cardiac fibroblasts(CFs) in rats. Methods The CFs taken from SD rats in 1 to 3 days were isolated by differential adhesion and observed under microscope. After cells were adherently grown to appropriate density, the cells were induced by TGF-β1 for modeling. The model cells were divided into the overexpression of ALKBH5 group infected by lentivirus and the negative control group for 24-48 hours. RT-qPCR was used to detect mRNA expression of ALKBH5, α-smooth muscle actin(α-SMA), type I collagen(Collagen Ⅰ) and proliferating cell nuclear antigen(PCNA). The expression of ALKBH5、α-SMA、Collagen Ⅰ and PCNA were assayed by Western blot. The cell proliferation activity was tested by CCK-8 assay and EdU. Results Compared with the control group, the protein and mRNA of ALKBH5 were reduced in the model group active by TGF-β1. Meanwhile, the biomarkers of activation, such as PCNA,α-SMA and Collagen Ⅰ, increased significantly. Besides, the protein and mRNA of PCNA、α-SMA and Collagen Ⅰ were lower in overexpression of ALKBH5 group than those of the negative control group. CCK-8 assay and EdU suggested that the proliferation viability of CFs was reduced evidently in overexpression of ALKBH5 group, compared with the negative control group. Conclusion Overexpression of ALKBH5 can inhibit the proliferation of CFs, suggesting that ALKBH5 may be a key regulatory point in the development of myocardial fibrosis.