Objective To exert different substrate strain stimulation on rabbit myofibroblasts (MFs) (proto-) at different differentiation stages so as to study the variation of α-SMA expression. MethodsBy implanting heterologous protein into rabbits’ peritoneal cavity, 5 d and 7 d MFs (proto-) were obtained and cultured on the elastic substrates. Strain of 0%-3% and 3%-6% as well as strain of 0%-6% were applied by substrate static stretch. Results α-SMA expression was not found in 3 d MFs (proto-), while α-SMA expression could be detected in 5-7 d MFs (proto-), and α-SMA expression was significantly enhanced in 10-15 d MFs (proto-) (P<0.01). Under the two loading modes, α-SMA expression was both significantly improved, and higher α-SMA expression was presented under sectioned loading. Conclusionsα-SMA expression of MFs（proto-） is increased after the substrate static stretch in response to different loading modes, which suggests that mechanical factors can play an important role in the process of MFs differentiation and wound healing response.