Vasoactive intestinal peptide (VIP) enhances angiogenesis in rats with focal cerebral ischemia. In the present study, we investigated the molecular mechanism of the proangiogenic action of VIP using an in vitro ischemic model, in which rat brain microvascular endothelial cells (RBMECs) are subjected to oxygen and glucose deprivation (OGD). Western blotting and immunocytochemistry were carried out to examine the expression of VIP receptors and vascular endothelial growth factor (VEGF) in cultured RBMECs. The cell proliferation was assessed by the MTT assay. Cyclic adenosine monophosphate (cAMP) and VEGF levels were measured by using the enzyme-linked immunosorbent assay. The cultured RBMECs expressed VPAC1, VPAC2 and PAC1 receptors. Treatment with VIP significantly promoted the proliferation of RBMECs and increased OGD-induced expression of VEGF, and this effect was antagonized by the VPAC receptor antagonist VIP6-28 and VEGF antibody. VIP significantly increased contents of cAMP in RBMECs and VEGF in the culture medium. The VIP-induced VEGF production was blocked by H89, a protein kinase A (PKA) inhibitor. These data suggest that treatment with VIP promotes VEGF-mediated endothelial cell proliferation after ischemic insult in vitro, and this effect appears to be initiated by the VPAC receptors leading to activation of the cAMP/PKA pathway.