AIM: To investigate the mechanism underlying the loss of responsiveness to anti-vascular endothelial growth factor (VEGF) treatment after repeated injections for choroidal neovascularization, VEGF and VEGF receptor (VEGFR) expressions were evaluated following repeated bevacizumab treatments in hypoxic human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: HUVECs were incubated under hypoxic conditions in two media of different bevacizumab concentrations (1.0 or 2.5 mg/mL) for 17h, and then in a new medium without bevacizumab for 7h. This procedure was repeated twice more. A culture with an identical volume of excipients served as the control. Cytotoxicity and cell proliferation were assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and Ki-67 assays, respectively. Levels of VEGF and VEGFR were assessed using enzyme-linked immunosorbent assay and Western blot respectively. RESULTS: Cytotoxic effects were not reported for either bevacizumab concentration. Cell proliferation was not reduced after anti-VEGF treatments. VEGF level after single treatment was significantly higher than that of the control and after repeated treatments. Phosphorylated VEGFR-2 expression increased significantly after single and repeated bevacizumab treatments compared with the control. The 1.0 mg/mL bevacizumab induced significantly higher expressions of VEGFR-2 than the 2.5 mg/mL in single and repeated treatment groups. CONCLUSION: Bevacizumab treatment of HUVECs elevated VEGFR expression in both single and repeated treatments, indicating a mechanism for the reduced efficacy of anti-VEGF therapy in ocular neovascular disorders.