Ischemic heart disease is a common cause of mortality worldwide. Sitagliptin is a new anti-diabetic drug acting as dipeptidyl peptidase-4 (DPP-4) inhibitor. The study investigated the ability of sitagliptin to prevent pathological changes of isoproterenol- (ISO-) induced myocardial injury in rats. The role of nitric oxide (NO) was also reported. Rats were assorted into six groups (n = 7) and treated for 12 days. Group 1: normal control, received normal saline. Group 2, sitagliptin control, received sitagliptin (10 mg/kg, orally). Group 3, ISO group, received isoproterenol (ISO) (100 mg/kg, i.p.). Group 4, sitagliptin + ISO, co-treated with sitagliptin plus ISO. Group 5, L-NNA + ISO, co-treated with L-N-nitro arginine (L-NNA) (25 mg/kg, orally) plus ISO. Group 6, sitagliptin + L-NNA + ISO, co-treated with sitagliptin plus ISO plus L-NNA. Blood glucose, serum creatine kinase-MB (CK-MB, and cardiac tissue parameters of oxidative stress parameters and NO, along with histopathological examination, and immunohistochemical study of inducible NO synthase (iNOS) expression were done. The results showed that sitagliptin caused a significant reduction in CK-MB, and attenuated histopathological damage-induced by ISO. Its effect was associated with a significant decrease in oxidative stress parameters, NO contents as well as by a significant decrease in the expression of iNOS in cardiac tissue. The protective effect of sitagliptin was abrogated by coadministartion of L-NNA, a selective inhibitor of both endothelial NOS (eNOS) and neuronal NOS (nNOS). In conclusion, sitagliptin ameliorates ISO-induced myocardial injury via antioxidant effects and modulation of NOS enzymes.