To explore the protective effect of N(2)-L-alanyl-L-glutamine (NLAG) on myocardial ischemia-reperfusion injury (IRI), and observe the influence of NLAG on the Janus activated kinase signal transducer 2 and activator of transcription 3 (JAK2/STAT3) signaling pathway‑associated molecules. Wistar rats were randomly divided into three groups: Sham, IRI and NLAG. In the IRI rat model, the cardiac hemodynamics, the maximum rate of left ventricular pressure (+dP/dtmax) and the left ventricular end‑diastolic pressure (LVDP) were recorded. Hematoxylin‑eosin and Masson staining were used to detect myocardial histological changes. The levels of plasma interleukin (IL)‑1β and ‑6, tumor necrosis factor (TNF)‑α, lactase dehydrogenase (LDH), troponin (cTn)I, creatine kinase (CK), heart type fatty acid binding protein (hFABP), malondialdehyde (MDA) and succinate dehydrogenase (SDH) were determined with ELISA. The protein expression levels of B‑cell lymphoma (Bcl)‑2, Bcl2‑associated X protein (Bax), Caspase‑3, JAK2, phosphorylated (p)‑JAK2, STAT3 and p‑STAT3 were detected by western blot analysis. The IRI model demonstrated notable myocardial injury; myocardial cells were arranged disorderly with some nuclei disappearing, and cardiac muscular fibers were degenerated. Following 60 min of reperfusion, LVDP, HR and +dP/dtmax were 31.3±4.53 mmHg, 239.17±8.45 beats/min and 615.17 mmHg/sec, respectively. Compared with the Sham group, the levels of LDH, cTnI, CK, hFABP release, inflammatory factors (IL‑1β, IL‑6 and TNF‑α) and oxygen free radical (MDA and SDH) levels were increased in the IRI group. In the NLAG group, myocardial injury was improved, the concentrations of LDH, cTnI, CK, hFABP, IL‑1β, IL‑6, TNF‑α, MDA were decreased, and SDH release was increased compared with the IRI group. In addition, NLAG significantly increased Bcl‑2, JAK2, p‑JAK2, STAT3 and p‑STAT3 protein expression, and decreased Bax protein expression compared with the IRI group. In conclusion, myocardial ischemia‑reperfusion can lead to myocardial cell apoptosis and myocardial injury and NLAG attenuates the IRI‑induced mitochondrial oxidative stress injury and apoptosis by activating the JAK2/STAT3 signaling pathway, thus exerting protective effects against IRI.