Proliferation and migration of Cardiac Fibroblasts (CFBs) are important in early stage of myocardial fibrosis. The purpose of the present study was to investigate the effect of CXCR4 antagonist AMD3100 treatment on transforming growth factor (TGF)β1induced CFBs abnormal proliferation and migration, and to elucidate the underlying mechanisms. The cell proliferation was evaluated by a MTT assay. Cell transwell migration assay and wound scratch assay were used to detect the cells migration. Measurement of ROS levels were determined by the DCF-DA dye. TGFβ1 stimulation in CFBs resulted in increased proliferation, migration and ROS generation. In conclusion, the present study revealed that AMD3100 treatment inhibited CFBs proliferation and migration induced by TGFβ1, at least in part through suppression of ROS signaling.