Scope: In this study, we focus on the effects of n-3 polyunsaturated fatty acids (PUFAs) on tunicamycin-, streptozotocin-, or high fat diet (HFD)-induced beta-cell damage and dysfunction. Materials and methods: Pretreatment with n-3 PUFAs protected RINm5F cells and mouse islets against tunicamycin- induced beta-cell damage through suppression of ER stress and apoptosis induction. This protective effect of n-3 PUFAs on beta-cells was further demonstrated by the normalization of insulin secretion in response to glucose in tunicamycin- treated islets. In multiple low-dose streptozotocin- induced diabetes models, fat-1 mice, which endogenously synthesize n-3 PUFAs from n-6 PUFAs, were fully resistant to the development of diabetes, with normal islet morphology, high insulin immunoreactivity, and decreased apoptotic cells. In HFD-induced diabetes models, fat-1 mice also exhibited improved glucose tolerance and functional beta-cell mass. In both diabetes models, we observed an attenuation of ER stress in fat-1 mice. Interestingly, n-3 PUFAs attenuated the nuclear translocation of lipogenic transcription factors sterol regulatory element-binding protein-1 (SREBP-1) and C/EBP beta, induced by tunicamycin or HFD, suggesting that n-3 PUFAs suppress ER stress via modulation of SREBP-1 and C/EBP beta. Conclusion: Together, these results suggest that n-3 PUFAs block ER stress, thus protecting beta cells against diabetogenic insult; therefore, dietary supplementation of n-3 PUFAs has therapeutic potential for the preservation of functional beta-cell mass.