Diabetic cardiomyopathy and nephropathy induce endoplasmic reticulum stress (ERS) and ERS-initiated apoptosis. The primary function of 14-3-3 protein is to inhibit apoptosis, but the roles of this protein in protecting against cardiac ERS and apoptosis in the diabetic heart are largely unknown. In this study, we investigated the in vivo role of 14-3-3 protein in diabetic ERS and apoptosis using streptozotocin (STZ)-induced transgenic mice that showed cardiac-specific expression of a dominant negative (DN) 14-3-3eta protein mutant. The expression levels of cardiac glucose-regulated protein (GRP) 78, inositol-requiring enzyme (Ire) 1alpha, and tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 2 protein were significantly increased in the diabetic DN 14-3-3eta mice compared with the diabetic wild-type. Moreover, cardiac apoptosis and the expression of CCAAT/enhancer binding protein homology protein (CHOP), caspase-12, and cleaved caspase-12 protein were significantly increased in the diabetic DN 14-3-3eta mice. In conclusion, partial depletion of 14-3-3 protein in the diabetic heart exacerbates cardiac ERS and activates ERS-induced apoptosis pathways, at least in part, through the regulation of CHOP and caspase-12 via the Ire1alpha/TRAF2 pathway. The enhancement of 14-3-3 protein expression can be used as a novel protective therapy against ERS and ERS-initiated apoptosis in the diabetic heart.