Endoplasmic reticulum (ER) stress is key in the development of cardiovascular diseases. However, there is a lack of a systemic ER stress‑induced cardiomyocyte apoptosis model. In the present study, primary cultured neonatal rat cardiomyocytes were exposed to tunicamycin. Cell viability was determined by an MTT assay, and cell damage was detected by a lactose dehydrogenase assay. Flow cytometry was used and the activity of caspase‑3 was analyzed in order to measure apoptosis. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to examine the expression of glucose‑regulated protein 78‑kDa (GRP78) and C/EBP homologous protein (CHOP). As a result, tunicamycin significantly increased cardiomyocyte injury, which occurred in a time- and concentration‑dependent manner. In addition, tunicamycin treatment resulted in apoptosis of cardiomyocytes. Molecularly, tunicamycin (100 ng/ml) increased the levels of GRP78 and CHOP 6 h after administration. In addition, GRP78 and CHOP reached maximum mRNA and protein levels 24 h after administration. In conclusion, the results implicate that the tunicamycin‑induced ER stress‑induced apoptotic model was successfully constructed in cultured neonatal rat cardiomyocytes. A 100 ng/ml concentration of tunicamycin was selected, and MTT, LDH release and flow cytometry assay was at 72 h. In addition, GRP78 and GRP94 were detected 24 h following administration. The results of the present study indicate a novel experimental basis for the investigation of ERS-induced cardiac apoptosis.