The beneficial effects of fish oil consumption on glucose metabolism have been generally reported. However, the mechanism underlying the fish oil-induced protective effects against insulin resistance remains unclear. Endoplasmic reticulum (ER) stress is recognized as an important contributor to insulin resistance. The aim of this study is to evaluate whether fish oil supplementation reduces ER stress and ameliorates insulin resistance in diet-induced obese mice, and to investigate the molecular mechanism of fish oil-induced benefits on ER stress. C57BL/6J mice were fed one of the following diets for 12 weeks: the low-fat diet (LFD), the high-fat diet (HFD) or the fish oil-supplemented high-fat diet (FOD). Fish oil supplementation led to lower blood glucose, better glucose tolerance and improved insulin sensitivity in high-fat diet-induced obese mice. Importantly, fish oil administration inhibited high-fat feeding-induced ER stress and reduced adipose tissue dysfunction. The fish oil-induced improvements were accompanied by the elevation of phosphorylated AMP-activated protein kinase (AMPK) expression in white adipose tissue. Correspondingly, the results of in vitro experiments showed that docosahexaenoic acid (DHA), the main n-3 polyunsaturated fatty acid (PUFA) in the fish oil used in the study, led to a dose-dependent increase in AMPK phosphorylation and suppressed palmitic acid (PA)-triggered ER stress in differentiated 3T3-L1 adipocytes. Furthermore, AMPK inhibitor (compound C) treatment largely blocked the effects of DHA to inhibit PA-induced ER stress. Our data indicate that n-3 PUFAs suppress ER stress in adipocytes through AMPK activation, and may thereby exert protective effects against high-fat feeding-induced adipose tissue dysfunction and insulin resistance.