Arginase 2 (ARG2) is a mitochondrial enzyme that catalyzes hydrolysis of L-arginine into urea and L-ornithine. In the kidney, ARG2 is localized to the S3 segment of the proximal tubule. It has been shown that expression and activity of this enzyme are upregulated in a variety of renal pathologies, including ischemia-reperfusion (IR) injury. However, the (patho)physiological role of ARG2 in the renal tubule remains largely unknown.We addressed this question in mice with conditional knockout of Arg2 in renal tubular cells (Arg2 /Pax8-rtTA/LC1 or, cKO mice).We demonstrate that cKO mice exhibit impaired urea concentration and osmolality gradients along the corticomedullary axis. In a model of unilateral IR injury (UIRI) with an intact contralateral kidney, ischemia followed by 24 hours of reperfusion resulted in significantly more pronounced histological damage in ischemic kidneys from cKO mice compared to control and sham-operated mice. In parallel, UIRI-subjected cKO mice exhibited a broad range of renal functional abnormalities, including albuminuria and aminoaciduria. Fourteen days after UIRI, the cKO mice exhibited complex phenotype characterized by significantly lower body weight, increased plasma levels of early predictive markers of kidney disease progression (ADMA and SDMA), impaired mitochondrial function in the ischemic kidney but no difference in kidney fibrosis as compared to control mice.Collectively, these results establish the role of ARG2 in the formation of corticomedullary urea and osmolality gradients and suggest that this enzyme attenuates kidney damage in ischemia-reperfusion injury.