Glucagon-like peptide-1 (GLP-1) analogues are likely to exert cardioprotective effects via balancing the energy metabolism in cardiomyocytes following ischemic or hypoxic insults. The present study aimed to explore the protective effects and mechanism of exendin-4, a GLP-1 analogue, on cardiomyocyte glucose uptake using an in vitro model of hypoxia/reoxygenation (H/R) of H9c2 cardiomyocyte cells. Pre-treatment with exendin-4 (200 nM) prior to H/R increased the cell viability, decreased cell apoptosis, enhanced cardiomyocyte glucose uptake and increased the production of adenosine triphosphate. Exendin-4 also decreased the levels of lactate dehydrogenase and creatine kinase-MB in the culture medium. Furthermore, the activity of carnitine palmitoyltransferase-1 in the H9c2 cells was decreased, while the activity of phosphofructokinase-1 was increased following exendin-4 treatment. Moreover, pre-treatment with exendin-4 increased the expression of p38 mitogen-activated protein kinase (p38MAPK) γ and translocation of glucose transporter-1 in H9c2 cells subjected to H/R. However, these effects were attenuated by the p38MAPK inhibitors BIRB796 and SB203580. The results suggested that exendin-4 exerted significant cardioprotective effects against H/R-induced cell injury and restored the metabolic imbalance of cardiomyocytes by activating the p38MAPK signaling pathway in the H9c2 cell model. Importantly, p38MAPKγ, one subunit of p38MAPK, may have the most important function in this process. The results of the present study may be helpful in the development of novel drugs to treat patients with coronary heart disease.