Free fatty acids cause pancreatic beta-cell apoptosis and may contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. Eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation is an adaptive response to ER stress, and reductions in eIF2alpha phosphorylation trigger beta-cell failure. Salubrinal inhibits eIF2alpha dephosphorylation and has been proposed as a novel therapy for diabetes. The objective of the study was to examine whether salubrinal modulates human islet susceptibility to lipotoxicity. Human islets were treated with oleate or palmitate, alone or in combination with salubrinal, and examined for apoptosis, ultrastructure, and gene expression. Salubrinal enhanced signaling downstream of eIF2alpha and markedly induced the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein, but it did not induce the inositol requiring-1alpha or activating transcription factor 6 ER stress pathways. Salubrinal potentiated the deleterious effects of oleate and palmitate in human islets. This proapoptotic effect involved ER dilation and mitochondrial rounding and fragmentation. Excessive eIF2alpha phosphorylation is poorly tolerated by human islets and exacerbates fatty acid-induced apoptosis through ER and mitochondrial mechanisms. This should be taken into consideration when designing approaches to pharmacologically modulate the beta-cell ER stress response in type 2 diabetes.