Prolonged endoplasmic reticulum (ER) stress contributes to the apoptosis of cardiomyocytes, which leads to the development of diabetic cardiomyopathy. Previously, we reported that the granulocyte colony-stimulating factor (G-CSF) reduces the cardiomyocyte apoptosis in diabetic cardiomyopathy; however, the precise mechanisms associated with this process are not yet fully understood. Therefore, in this study, we investigated whether the mechanism of the anti-apoptotic effect of G-CSF was associated with ER stress in a rat model of diabetic cardiomyopathy. Diabetic cardiomyopathy was induced in rats using a high-fat diet combined with the administration of a low-dose of streptozotocin. Diabetic rats were treated with G-CSF or saline for 5 days. Cardiac function was evaluated using serial echocardiography before and 4 weeks after treatment. The rate of cardiomyocyte apoptosis and the expression levels of proteins related to ER stress, including glucose-regulated protein 78 (GRP78), caspase-9, and caspase-12 were analyzed in the cardiac tissue. G-CSF treatment significantly reduced cardiomyocyte apoptosis in the diabetic myocardium and downregulated the expression levels of these proteins in diabetic rats treated with low-dose streptozotocin when compared to that in rats treated with saline. In addition, G-CSF treatment significantly downregulated the expression levels of proteins related to ER stress, such as GRP78, inositol-requiring enzyme-1α (IRE-1α), and C/EBP homologous protein (CHOP) in H9c2 cells under high glucose (HG) conditions. Moreover, G-CSF treatment significantly improved the diastolic dysfunction in serial echocardiography assessments. In conclusion, the anti-apoptotic effect of G-CSF may be associated with the downregulation of ER stress.