Although previous studies have shown that γ-secretase inhibitors significantly suppress tumor growth via anti-angiogenesis, the mechanism involved in the regulation of tumor angiogenesis by γ-secretase inhibitors has not been clearly understood. The objective of this study was to investigate the regulation of vascular endothelial growth factor receptor (VEGFR) and endothelial nitric oxide synthase (eNOS) by a γ-secretase inhibitor in the H5V mouse microvascular endothelial cell line. H5V cells were cultured with different concentrations of the γ-secretase inhibitor DAPT for 48 h and with 100 μmol/l DAPT at different incubation times. Protein and mRNA expression of VEGFR-1, VEGFR-2, VEGFR-3 and eNOS was measured by Western blotting and real-time PCR, respectively. The VEGFR-2 kinase inhibitor was used to assess the role of VEGFR-2 in eNOS regulation. We found that the γ-secretase inhibitor DAPT increased protein and mRNA expression of VEGFR-2 and eNOS, but decreased VEGFR-1 expression and had no significant effect on VEGFR-3. Up-regulation of eNOS was blocked by the VEGFR-2 kinase inhibitor. In conclusion, the γ-secretase inhibitor enhances VEGFR-2 and eNOS expression, and the up-regulation of eNOS is dependent on an increase in VEGFR-2. Thus, we suggest that administration of the γ-secretase inhibitor be combined with disruption of eNOS or interruption of VEGF signaling, which may improve the anti-angiogenic efficacy in tumor treatments.