The endothelial barrier function is tightly controlled by a broad range of signaling cascades including nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway. It has been proposed that disturbances in NO and cGMP production could interfere with proper endothelial barrier function. In this study, we assessed the effect of interferon-gamma (IFN-γ), a pro-inflammatory cytokine, on NO and cGMP levels and examined the mechanisms by which NO and cGMP regulate the IFN-γ-mediated HUVECs hyperpermeability. The flux of fluorescein isothiocyanate-labeled dextran across cell monolayers was used to study the permeability of endothelial cells. Here, we found that IFN-γ significantly attenuated basal NO concentration and the increased NO levels supplied by a NO donor, sodium nitroprusside (SNP). Besides, application of IFN-γ also significantly attenuated both the basal cGMP concentration and the increased cGMP production donated by a cell permeable cGMP analogue, 8-bromo-cyclic GMP (8-Br-cGMP). In addition, exposure of the cell monolayer to IFN-γ significantly increased HUVECs basal permeability. However, L-NAME pretreatment did not suppress IFN-γ-induced HUVECs hyperpermeability. L-NAME pretreatment followed by SNP or SNP pretreatment partially reduced IFN-γ-induced HUVECs hyperpermeability. Pretreatment with a guanylate cyclase inhibitor, 6-anilino-5, 8-quinolinedione (LY83583), led to a further increase in IFN-γ-induced HUVECs hyperpermeability. The findings suggest that the mechanism underlying IFN-γ-induced increased HUVECs permeability is partly related to the inhibition of NO production.