Objective – To date, the biological function of FAM3A, a member of FAM3 gene family, remains unknown. This study aimed to determine the role of FAM3A in proliferation and migration of vascular smooth muscle cells (VSMCs). Methods and Results – Immunohistochemical staining revealed that FAM3A protein is expressed in tunica media of rodent and human arteries. FAM3A expression is reduced in tunica media of mouse femoral artery and rat carotid artery injuried by a guidewire or balloon, and in cultured rat VSMCs stimulated by FBS or PDGF-BB with increased expression of prostaglandin E receptor 2 (EP2). Adenoviral-mediated overexpression of FAM3A promotes proliferation and migration of VSMCs independent of FBS stimulation. FAM3A activates Akt via a PI3K-dependent manner, whereas it induces ERK1/2 activation independent of PI3K in VSMCs. FAM3A protein is located in mitochondrion of VSMCs, where it promotes ATP produce and secretion. Activation or overexpression of EP2 represses FAM3A expression with inhibited ATP produce and secretion in VSMCs. FAM3A-induced activation of Akt and ERK1/2 pathways, proliferation and migration of VSMCs is blocked by suramin, a broad-spectrum P2 receptor inhibitor, but not by PPADS, a specific P2X receptor inhibitor. Moreover, inhibition of phospholypase C (PLC) and inositol 1,4,5-triphosphate receptor (IP3R) also blocks FAM3A-induced activation of Akt and ERK1/2 pathways, and proliferation of VSMCs. Conclusion – FAM3A promotes proliferation and migration of VSMCs via P2Y receptor-mediated activation of Akt and ERK1/2 pathways. Repression of FAM3A expression by EP2 activation might be a protective mechanism against excessive neointima formation in injuried vessles.