Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) exert a protective role against retinal injuries, including diabetic macular edema (DME). The macular damage is induced by hyperglycemia, which damages vessels supplying blood to the retina and induces hypoxia. The microenvironmental changes stimulate the expression of hypoxia‐inducible factors (HIFs), which promote the choroidal endothelial cell transmigration across the retinal pigmented epithelium (RPE) into neurosensory retina, where they proliferate into new vessels under stimulation of the vascular endothelial growth factor (VEGF). In the present study, we have investigated whether PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF, and its receptors. In accord to our hypothesis, we have shown that both peptides are able to significantly reduce HIF‐1α and increase HIF‐3α expression in ARPE‐19 cells exposed to hyperglycemic/hypoxic insult. This effect is also related to a reduction of VEGF and its receptors expression. Moreover, both peptides also reduce the activation of p38 mitogen‐activated protein kinase (MAPK), a pro‐apoptotic signaling pathway, which is activated by VEGFR‐1 and 2 receptors. In conclusion, our study has further elucidated the protective role performed by PACAP and VIP, against the harmful combined effect of hyperglycemia/hypoxia characterizing the DME microenvironment. J. Cell. Physiol. 232: 1209–1215, 2017. © 2016 Wiley Periodicals, Inc. PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF, and its receptors; PACAP and VIP act against the harmful combined effect of hyperglycemia/hypoxia.