Atherosclerosis or vascular injury causes contractile smooth muscle cells (SMCs) to dedifferentiate and proliferate by re-entering the cell cycle, thus contributing to vascular pathology and dysfunction.There is increasing evidence that activation of the G-protein coupled estrogen receptor (GPER) protects against cardiovascular disease, but it is unknown whether GPER plays a role in coronary artery smooth muscle cell (CASMC) proliferation and differentiation.The objective of our study was to understand the functional role of GPER on CASMC proliferation and differentiation in coronary arteries from humans and from normal and diabetic swine.We found that G-1 (GPER agonist) inhibited both human and porcine CASMC proliferation in a concentration-(10-8 to 10-5 M) and time-dependent manner.Flow cytometry revealed that G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 induces cell proliferation by slowing down progression of the cell cycle.Further, G1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21.Our findings also suggested that these inhibitory effects of G-1 might involve decreased phosphorylation of ERK1/2 and Akt.In addition, G-1 induced SMC differentiation marker protein alphaactinexpression, consistent with Western blotting analysis demonstrating increased expression of both SM22-alpha and alpha-actin after G-1 treatment.These findings suggest that G-1 induces differentiation of growing VSMC.Lastly,we detected increased expression of GPER and cell proliferation marker PCNA in coronary arteries from diabetic swine, suggesting a correlation between GPER and PCNA expression.We conclude that GPER activation inhibits CASMC proliferation by slowing cell cycle progression via inhibiting ERK1/2 and Akt activity, and suggest that GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.In contrast, GPER expression correlates with VSMC proliferation in diabetic mellitus.