Hyperglycemia is implicated both in micro- and macro-vascular complications in diabetes mellitus. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two known nonclassic regulators of angiogenesis, although their biological role on endothelial cell proliferation remains poorly defined. In the present study we hypothesized that either peptides might play an inhibitory role on hyperglycemia-induced cell growth. To this end, we investigated the effect of both PACAP and VIP on cell proliferation in murine microvascular endothelial cells (H5V) cultured both under euglycemic and hyperglycemic conditions (5 and 25 mM glucose, respectively) for 24, 48 h, 7 and 15 days. Results demonstrated that high glucose treatment induced a time-dependent increase in cell viability after 48 h (p<0.05), which was much more evident after 7 and 15 days (p<0.001). Similar effects were observed in cell proliferation, although significant changes were obtained after prolonged exposures to high glucose (7 and 15 days; p<0.001). The proliferative response to the glucose-enriched environment was correlated to changes in the expression of PAC1 and, to a minor extent, to VPAC2, but not VPAC1 receptors, as measured by quantitative real-time PCR. These results were further confirmed by Western blot and immunofluorescence analyses. Interestingly, 10⁻⁷ M PACAP or VIP treatment significantly attenuated hyperglycemia-induced increase in cell viability and proliferation after 7 and 15 days. Taken together, our findings demonstrate that both PACAP and VIP peptides exert an inhibitory activity on hyperglycemia-induced endothelial cell proliferation, thus suggesting that the effect might be mediated by PAC1 and VPAC2 receptors.