Far red/near infrared light (NIR) is beneficial against cardiac ischemia and reperfusion injury (I/R), although the exact underlying mechanism is unknown. Previously we established that NIR enhanced the cardioprotective effect of nitrite in the rabbit heart. Furthermore, we observed that the nitrosyl myoglobin (MbNO) level in ischemic tissue decreased upon irradiation of the heart. Our hypothesis was that protection against I/R is dependent on nitric oxide (NO)-release from heme-proteins, and remains present during diabetes. When mice were subjected to I/R NIR (660 nm) applied during the beginning of reperfusion reduced infarct size dose dependently compared to untreated animals. Similarly, the isolated (Langendorff) heart model resulted in sustained left ventricular diastolic pressure after I/R in NIR-treated hearts. NIRinduced protection was preserved in a diabetic mouse model (db/db) and during acute hyperglycemia. NIR liberated NO from nitrosyl hemoglobin (HbNO) and MbNO as well as from HbNO isolated from the blood of diabetic animals. In the Langendorff model, after application of the nitrosylated form of a hemoglobin-based oxygen carrier as an NO donor NIR induced an increase in NADH level, suggesting a mild inhibition of mitochondrial respiration by NO during reperfusion. Taken together, NIR applied during reperfusion protects the myocardium against I/R in a NO-dependent and mitochondrion-targeted manner. This unique mechanism is conserved under diabetic conditions where other protective strategies fail.