H₂O₂ mediates autocrine and paracrine signaling in the vasculature and can propagate endothelial dysfunction. However, it is not clear how endothelial cells withstand H₂O₂ exposure and promote H₂O₂-induced vascular remodeling. To understand the innate ability of endothelial cells for sustaining excess H₂O₂ exposure, we investigated the genotypic and functional regulation of redox systems in primary HUVECs following an H₂O₂ treatment.Primary HUVECs were exposed to transient H₂O₂ exposure and consistent H₂O₂ exposure. Following H₂O₂ treatments for 24, 48 and 72h, we measured O₂− production, mitochondrial membrane polarization (MMP), and gene expressions of pro-oxidative enzymes, peroxidase enzymes, and cytoprotective intermediates.Our results showed that the 24h H₂O₂ exposure significantly increased O₂− levels, hyperpolarized MMP, and downregulated CAT, GPX1, TXNRD1, NFE2L2, ASK1, and ATF2 gene expression in HUVECs. At 72h, HUVECs in both treatment conditions were shown to adapt to reduce O₂− levels and normalize MMP. An upregulation of GPX1, TXNRD1, and HMOX1 gene expression and a recovery of NFE2L2 and PRDX1 gene expression to control levels were observed in both consistent and transient treatments at 48 and 72h.The response of endothelial cells to excess levels of H₂O₂ involves a complex interaction amongst O₂− levels, mitochondrial membrane polarization and anti- and pro-oxidant gene regulation. As a part of this response, HUVECs induce cytoprotective mechanisms including the expression of peroxidase and antioxidant enzymes along with the downregulation of pro-apoptotic genes. This adaptation assists HUVECs to withstand subsequent exposures to H₂O₂.