The present study investigated the possible involvement of nitric oxide/soluble guanylyl cyclase (NO/sGC) pathway in ascorbic acid (AA)-mediated protection against acute kidney injury (AKI) in rats. The rats were subjected to bilateral renal ischemia by occluding renal pedicles for 40 min followed by reperfusion for 24 h. The AKI was assessed in terms of measuring creatinine clearance (CrCl), blood urea nitrogen (BUN), plasma uric acid, potassium level, fractional excretion of sodium (FeNa), and microproteinuria. The NO level and oxidative stress in renal tissues were assessed by measuring myeloperoxidase activity, thiobarbituric acid reactive substances, superoxide anion generation, and reduced glutathione level. AA (50 and 100 mg/kg, p.o.) was administered for 3 days before subjecting rats to AKI. In separate groups, the nitric oxide synthase inhibitor, L-NAME (20 mg/kg, i.p.) and sGC inhibitor, methylene blue (50 mg/kg, i.p.) was administered prior to AA treatment in rats. The significant decrease in CrCl and increase in BUN, plasma uric acid, potassium, FeNa, microproteinuria, and oxidative stress in renal tissues demonstrated ischemia-reperfusion-induced AKI in rats. The AA treatment ameliorated ischemia-reperfusion-induced AKI along with the increase in renal NO level. The pretreatment with L-NAME and methylene blue abolished protective effect of AA. It is concluded that AA protects against ischemia-reperfusion-induced AKI. Moreover, the NO/sGC pathway finds its definite involvement in AA-mediated reno-protective effect.