Vascular endothelial growth factor (VEGF)-A induces endothelial hyperpermeability, but the molecular pathways remain incompletely understood. Endothelial nitric oxide synthase (eNOS) regulates acute effects of VEGF-A on permeability of endothelial cells (ECs), but it remains unknown whether and how eNOS regulates late effects of VEGF-A-induced hyperpermeability. Here we show that VEGF-A induces hyperpermeability via eNOS-dependent and eNOS-independent mechanisms at 2 days after VEGF-A stimulation. Silencing of expression of the eNOS gene (NOS3) reduced VEGF-A-induced permeability for dextran (70 kDa) and 766 Da-tracer in human dermal microvascular ECs (HDMVECs), but not in human retinal microvascular ECs (HRECs) and human umbilical vein ECs (HUVECs). However, silencing of NOS3 expression in HRECs increased permeability to dextran, BSA and 766 Da-tracer in the absence of VEGF-A stimulation, suggesting a barrier-protective function of eNOS. We also investigated how silencing of NOS3 expression regulates the expression of permeability-related transcripts, and found that NOS3 silencing downregulates the expression of PLVAP, a molecule associated with trans-endothelial transport via caveolae, in HDMVECs and HUVECs, but not in HRECs. Our findings underscore the complexity of VEGF-A-induced permeability pathways in ECs and the role of eNOS therein, and demonstrate that different pathways are activated depending on the EC phenotype.