Exendin-4, a glucagon-like peptide-1 receptor agonist, demonstrated cytoprotective actions beyond glycemic control in recent studies. The aims of the present study were to investigate the effects of exendin-4 on high glucose (HG)-induced cardiomyocyte apoptosis and the possible mechanisms. Rat cardiomyocytes were divided into 3 groups: normal glucose group (NG group), HG group and HG +exendin-4 group (HG+Ex Group). Cardiomyocyte apoptosis was evaluated by double-staining with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and flow cytometry. Intracellular reactive oxygen species (ROS) production was detected by 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) incubation and fluorescence microscopy. LY294002 (LY), a phosphoinositide 3-kinase (PI3K) pathway inhibitor, was added to the medium of the HG+Ex+LY Group for further western blot analysis. The proteins analyzed involved oxidative stress-associated proteins, heme oxygenase-1 (HO-1) and nuclear factor E2-related factor 2 (Nrf-2), and apoptosis-associated proteins, caspase-3, Bax/B-cell lymphoma 2 (Bcl-2) and p-AKT/AKT. HG treatment induced cardiomyocyte apoptosis (P = 0.00) and clearly upregulated ROS production (P = 0.00); exendin-4 co-incubation also ameliorated cardiomyocyte apoptosis (P = 0.004) and decreased ROS (P = 0.00) level significantly. HO-1 and Nrf-2 protein expression levels decreased significantly in the HG group (P < 0.05), but the levels were elevated by exendin-4 intervention (P < 0.05). Furthermore, exendin-4 attenuated HG-induced higher protein expression, including cleaved caspase-3 and Bax, increased the expression of Bcl-2 protein (P < 0.05). However, these impacts of exendin-4 were counteracted significantly by co-incubation with LY294002. In addition, exendin-4 ameliorated HG-induced p-AKT/AKT lower expression, and this impact was also suppressed by LY294002. Exendin-4 ameliorates HG-induced cardiomyocyte apoptosis, and the mechanisms may involve anti-oxidative stress via the HO-1/Nrf-2 system, as well as intervention of the PI3K/AKT signaling pathway.