The arterial system is subjected to cyclic strain because of periodic alterations in blood pressure, but the effects of frequency of cyclic strain on arterial smooth muscle cells (SMCs) remain unclear. Here, we investigated the potential role of the cyclic strain frequency in regulating SMC alignment using an in vitro model. Aortic SMCs were subject to cyclic strain at one elongation but at various frequencies using a Flexercell Tension Plus system. It was found that the angle information entropy, the activation of integrin-beta1, p38 MAPK, and F/G actin ratio of filaments were all changed in a frequency-dependent manner, which was consistent with SMC alignment under cyclic strain with various frequencies. A treatment with anti-integrin-beta1 antibody, SB202190, or cytochalasin D inhibited the cyclic strain frequency-dependent SMC alignment. These observations suggested that the frequency of cyclic strain plays a role in regulating the alignment of vascular SMCs in an intact actin filament-dependent manner, and cyclic strain at 1.25 Hz was the most effective frequency influencing SMC alignment. Furthermore, integrin-beta1 and p38 MAPK possibly mediated cyclic strain frequency-dependent SMC alignment.