Endothelial-to-mesenchymal transition (EndoMT) has recently emerged as a potentially important contributor in promoting fibrosis in chronic kidney disease. However, little is known about the role and molecular basis of its involvement in hypertensive renal injury. Here, we aim to determine the role of SIRT (sirtuin) 3 on EndoMT in hypertensive renal injury and to explore its underlying mechanisms. We found that SIRT3 expression was significantly reduced in Ang II (angiotensin II)–induced hypertensive model, accompanied with induction of EndoMT and increased reactive oxygen species and renal fibrosis. In SIRT3−/− (SIRT3 knockout) mice subjected to Ang II infusion, renal dysfunction was aggravated with an increased EndoMT and reactive oxygen species level, whereas in SIRT3-TgEC (SIRT3 endothelial cell–specific transgenic) mice, the Ang II-induced renal fibrosis and EndoMT and oxidative stress were ameliorated. With primary mouse glomerular endothelial cells, we confirmed that Ang II treatment initiated EndoMT and decreased catalase expression, which were suppressed by SIRT3 overexpression. Using immunoprecipitation, luciferase, and chromatin immunoprecipitation assay, we demonstrated that SIRT3-mediated deacetylation and nuclear localization of Foxo3a (forkhead box O3a) resulted in activated Foxo3a-dependent catalase expression. Moreover, Foxo3a knockdown abolished SIRT3-mediated suppression of EndoMT. In conclusion, these results established the SIRT3-Foxo3a-catalase pathway as a critical factor in the maintenance of endothelial homeostasis and point to an important role of EndoMT in the vascular pathology of renal fibrosis, which may provide a new therapeutic target to impede the progression of hypertensive renal injury.