Aim To investigate the effect of microRNA miR-25-3p on fibrosis-related gene expression in cardiac fibroblasts and mechanism involved. Methods A mouse model of cardiac fibrosis induced by angiotensin Ⅱ (AngⅡ) infusion was constructed, and the differentially expressed miRNAs in the fibrotic mouse myocardium were detected by miRNA chip array. Primary isolation and culture of C57BL/6 mouse cardiac fibroblasts (mCF) were performed, and a cell model of myocardial fibrosis was established based on AngⅡ-treated mCF. The effect of miR-25-3p mimic on the expression of fibrosis-related genes in mCF was studied. The dual luciferase reporter gene assay was performed to verify the binding of miR-25-3p on the 3′-untranslated region (3′UTR) of the B-cell translocation gene 2 (BTG2) gene. Actinomycin D experiment was performed to detect the effect of BTG2 on the stability of superoxide dismutase 2 (SOD2) mRNA in mCF. Results Expression of miR-25-3p was increased in the myocardium of AngⅡ-infused mice and AngⅡ-treated mCF. miR-25-3p could enhance the expression of fibrosis-related genes, including collagen type Ⅰ alpha 1 chain (COL1A1), collagen type Ⅲ alpha 1 chain(COL3A1) and α-smooth muscle actin(α-SMA) in mCF. BTG2 was confirmed to be a target gene of miR-25-3p, and BTG2 mediated the effect of miR-25-3p on promoting fibrosis-related gene expression in mCF. In addition, BTG2 could enhance the expression of SOD2 by increasing the stability of SOD2 mRNA in mCF. Conclusion MiR-25-3p inhibited the level of SOD2 through targeting BTG2 in mCF, resulting in enhancing fibrosis-related gene expression and promoting myocardial fibrosis.