Endoplasmic reticulum (ER) stress (ERS) is involved in various cardiovascular diseases. Our previous study verified that ERS took part in the development of cardiac hypertrophy; however, its mechanism is still unclear. This study aimed to investigate the roles of the calcineurin (CaN) signal pathway in hypertrophy induced by the ERS inductor thapsigargin (TG) in neonatal cardiomyocytes from Sprague-Dawley rats. Investigation of ER chaperone expression, ER staining, and calreticulin immunofluorescence were used to detect the ERS response. mRNA expression of atrial natriuretic peptide and brain natriuretic peptide, total protein synthesis rate, and cell surface area were used to evaluate cardiac hypertrophy induced by TG. TG induced a significant ERS response along with hypertrophy in a dose- and time-dependent manner in cardiomyocytes, which was verified by treatment with tunicamycin, another ERS inducer. Furthermore, TG induced a significant elevation of the intracellular Ca(2+) level, CaN activation, and myocyte enhancer factor 2c (MEF2c) expression in a dose- and time-dependent manner in cardiomyocytes. Cyclosporine A, a CaN inhibitor, markedly suppressed MEF2c nuclear translocation and inhibited TG-induced hypertrophy. These results demonstrate that ERS induces cardiac hypertrophy and that the CaN-MEF2c pathway is involved in ERS-induced hypertrophy in cardiomyocytes.