Aim To explore possible action mechanism of mitogen-activated protein kinase (MAPK) signal transduction pathway on chemotaxis of cardiac fibroblasts (CF) induced by transforming growth factor-β1 (TGF-β1). Methods The CF of neonatal SD rats were cultured. Cell were randomly divided into blank control group, TGF-β1 group, c-Jun N-terminal kinase (JNK) inhibitor (SP600125 10 μmol/L) group, P38MAPK inhibitor (SB203580 10 μmol/L) group and extracellular signal-regulated protein kinase (ERK) inhibitor (U0126 10 μmol/L) group. Except blank control group, the other groups were given 10 μg/L TGF-β1 and corresponding inhibitors. Methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to detect cell viability of CF. Transwell chamber was applied to detect motor ability of CF. The collagen content was detected by hydroxyproline kit. The enzyme-linked immunosorbent assay (ELISA) was applied to detect levels of monocyte chemotactic protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) in CF. Western blot was applied to detect expression levels of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col-1) and matrix metalloproteinase-9 (MMP-9) in CF. Results TGF-β1 could significantly promote cell viability and chemotaxis of CF and collagen content. MAPK inhibitors could inhibit the proliferation and chemotaxis, and decrease collagen content (P＜0.05). ELISA results showed that MAPK signaling pathway inhibitors could significantly decrease increased MCP-1 and PAI-1 induced by TGF-β1 treatment (P＜0.05). Western blot results showed that MAPK signaling pathway inhibitors could significantly decrease increased expression levels of α-SMA, Col-1 and MMP-9 protein induced by TGF-β1 treatment (P＜0.05). Conclusion TGF-β1 may promote chemotaxis of CF by activating MAPK signaling pathway. The application of MAPK inhibitors can inhibit myocardial fibrosis to certain extent.