Objective: To investigate the functional role of endoplasmic reticulum stress (ERS) in eicosapentaenoic acid (EPA) attenuating myocardial apoptosis induced by palimitate (PAL). Methods: Cells were divided randomly into six groups: the blank control group, the PAL stimulated group, the EPA + PAL stimulated group, the thapsigargin + EPA + PAL-stimulated group, thapsigargin group, thapsigargin + EPA stimulated group. CCK-8 was used to test the cell viability, TUNEL staining was used to detect the apoptosis, and Western blot method was used to detect the expression of glucose-regulated protein 78 (GRP78), calreticulin (CRT), C/EBP homologous protein (CHOP), JNK and caspase-12. Results: PAL treatment resulted in a decrease of cell viability and an increase of cell apoptotic rates and led to a significant up-regulation of the expression of GRP78, CRT, CHOP, p-JNK and cleaved caspase-12 proteins. Compared with PAL treatment, pretreatment with EPA + PAL resulted in a significant decrease of the expression of GRP78, CRT, cleaved caspase-12, p-JNK and CHOP proteins (P<0.05), and decreased PAL-induced cardiomyocytes apoptosis from 43.9% to 24.07% (P<0.05). Cells were treated with thapsigargin, a specific activator of ERS; compared with EPA+PAL treatment, thapsigargin + EPA + PAL showed increased significantly of the expression of GRP78, cleaved caspase-12, p-JNK and CHOP proteins (P<0.05), down-regulated of cell viability, and enhanced apoptosis of cardiomyocyte cell. Furthermore, thapsigargin pretreatment obviously decreased cell viability and increase cell apoptosis compared with the control group, pretreatment with thapsigargin + EPA decreased thapsigargin-induced cardiomyocytes apoptosis. Conclusion: EPA could prevent from PAL-induced cardiomyocytes apoptosis through a restraining ERS and caspase dependent apoptotic pathway.