Phenotype transformation of vascular smooth muscle cells (VSMCs) has been reported to be directly influenced by the frequency of mechanical strain. This study explored the effects of different frequencies of mechanical strain on expression of phenotype marker h1-calponin and the possible mechanism. VSMCs were subjected to cyclic strains of 10% elongation at 1 and 2 Hz for 24 h by using a Flexercell strain unit. The protein expression of h1-calponin was assessed by Western blotting and the possible protein kinases involved were evaluated by their specific inhibitor or targeted siRNA 'knock-down.' The results showed that cyclic strains modulated the expressions of h1-calponin, phospho-p38, Rac and Rho-guanine nucleotide dissociation inhibitor alpha (Rho-GDI alpha) in nonlinear frequency-dependent manners. This nonlinear frequency-dependent change of h1-calponin expression could be blocked by a specific p38 inhibitor, SB202190. The changed expression of phospho-p38 induced by the frequencies of cyclic strain was reversed by targeted siRNA 'knock-down' of Rac, while enhanced by targeted siRNA 'knock-down' of Rho-GDI alpha. These results suggest that the frequency-dependent expression of h1-calponin under cyclic strain is mediated at least partly by the regulation of Rac and Rho-GDI alpha expression on the activation of p38 pathway.