Novel therapeutic interventions for preventing or attenuating kidney injury following ischemia/reperfusion injury (IRI) remain a focus of significant interest. Currently, there are no definitive therapeutic or preventive approaches available for ischemic acute kidney injury (AKI). Our objective is to determine (a) whether renal arginase activity or expression is increased in renal IRI and (b) whether arginase plays a role in development of renal IRI. The impact of arginase activity and expression on renal damage was evaluated in male C57BL/6J (WT) and arginase-2 deficient (Arg2^-/-) mice subjected to bilateral renal ischemia for 28min followed by reperfusion for 24h. ARG2 expression and arginase activity significantly increased following renal IRI, paralleling the increase in kidney injury. Pharmacological blockade or genetic deficiency of Arg2 conferred kidney protection in renal IRI. Arg2^-/- mice had significantly attenuated kidney injury and lower plasma creatinine and blood urea nitrogen levels after renal IRI. Blocking arginases using S-(2-boronoethyl)-L-cysteine (BEC) 18h before ischemia mimicked arginase deficiency by reducing kidney injury, histopathological changes and kidney injury marker-1 expression, renal apoptosis, kidney inflammatory cell recruitment and inflammatory cytokines, kidney oxidative stress, and increased kidney NO production and eNOS phosphorylation, kidney PPAR co-activator (PGC)-1α expression, mitochondrial ATP, and preserved kidney mitochondrial ultrastructure compared to vehicle-treated IRI mice. Importantly, BEC-treated eNOS-knockout mice failed to reduce BUN and creatinine following renal IRI. These findings indicate that arginase-2 plays a major role in renal IRI, via an eNOS-dependent mechanism and that blocking ARG2 activity or expression could be a novel therapeutic approach for prevention of AKI.