Aim To explore the effect of STIM1 knockdown on cell cycle of vascular smooth muscle cell. Methods Rat VSMC was isolated and primary cultured. Ad-si/rSTIM1 and Ad-hSTIM1 were transfected into VSMC. The protein of STIM1, p21 and pRb were measured by western blot, the cell cycle of VSMC was analyzed by flow cytometry. Results On 48 h after transfected Ad-si/rSTIM1, the expression of STIM1 protein was decreased significantly compared to that in Ad-hSTIM1-treated group (P<0.05). The percent of G0/G1 phase cell was significantly increased (P<0.05). The expression of p21 protein was increased and the expression of pRb protein was decreased (P<0.05). However, the cotransfection of Ad-hSTIM1 with Ad-si/rSTIM1 restored these responses. Conclusions STIM1 knockdown up-regulates the expression of p21 and down-regulates the expression of pRb, which inhibits cell to progress into S phase. Stim1 maybe have a key role in the vascular smooth muscle cell proliferation.