Abstract: AIM: To investigate the effect of iNOS and phosphorylation of ERK1/2 on 17β-estradiol (E2)-induced inhibition of vascular smooth muscle cells (VSMCs). METHODS: In cultured VSMCs, proliferation was determined by MTT assay, and the expression of iNOS and ERK1/2 phosphorylation were measured by Western blot. RESULTS: The measurement showed that exposure to fetal calf serum (FCS) for 24 h increased the proliferation of VSMCs. Pretreatment with E2 for 24 h inhibited VSMC proliferation under growth-stimulating (10% FCS) condition. Western blot showed that FCS inhibited expression of iNOS and increased expression of ERK1/2 phosphorylation in VSMCs. Pretreatment with E2 for 24 h partly reversed this effect. Western blot results further proved that pretreatment with iNOS inhibitor, L-NAME, partly reversed the increase of ERK1/2 phosphorylation induced by FCS. CONCLUSION: Our results demonstrate that 17β-estradiol increases iNOS expression and decreases ERK1/2 phosphorylation, leading to inhibition of VSMC proliferation.