BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation is involved in many types of arterial diseases, including neointima hyperplasia, in which Ca has been recognized as a key player. However, the physiological role of Ca release via inositol 1,4,5-trisphosphate receptors (IPRs) from endoplasmic reticulum in regulating VSMC proliferation has not been well determined. METHODS AND RESULTS: Both in vitro cell culture models and in vivo mouse models were generated to investigate the role of IPRs in regulating VSMC proliferation. Expression of all 3 IPR subtypes was increased in cultured VSMCs upon platelet-derived growth factor-BB and FBS stimulation as well as in the left carotid artery undergoing intimal thickening after vascular occlusion. Genetic ablation of all 3 IPR subtypes abolished endoplasmic reticulum Ca release in cultured VSMCs, significantly reduced cell proliferation induced by platelet-derived growth factor-BB and FBS stimulation, and also decreased cell migration of VSMCs. Furthermore, smooth muscle-specific deletion of all IPR subtypes in adult mice dramatically attenuated neointima formation induced by left carotid artery ligation, accompanied by significant decreases in cell proliferation and matrix metalloproteinase-9 expression in injured vessels. Mechanistically, IPR-mediated Ca release may activate cAMP response element-binding protein, a key player in controlling VSMC proliferation, via Ca/calmodulin-dependent protein kinase II and Akt. Loss of IPRs suppressed cAMP response element-binding protein phosphorylation at Ser133 in both cultured VSMCs and injured vessels, whereas application of Ca permeable ionophore, ionomycin, can reverse cAMP response element-binding protein phosphorylation in IPR triple knockout VSMCs. CONCLUSIONS: Our results demonstrated an essential role of IPR-mediated Ca release from endoplasmic reticulum in regulating cAMP response element-binding protein activation, VSMC proliferation, and neointima formation in mouse arteries.