n-3 polyunsaturated fatty acids (PUFA) can exert beneficial effects on glucose homeostasis, especially in obese rodents. Gut incretin hormones regulate glucose and lipid homeostasis, but their involvement in the above effects is not entirely clear. This study aims to assess the effects of chronic n-3 PUFA administration on the insulin and incretin responses in C57BL/6N obese male mice subjected to oral glucose tolerance test (oGTT) after 8 weeks of feeding a corn-oil-based high-fat diet (cHF). The weight gain and adiposity were partially reduced in mice fed cHF in which some of the corn oil was replaced with n-3 PUFA concentrate containing ∼60% DHA and EPA in a 3 : 1 ratio. In addition, these mice had improved glucose tolerance, which was consistent with an increased insulin response to oral glucose and plasma glucagon-like peptide-1 (GLP-1) levels. While the stimulatory effects of n-3 PUFA on GLP-1 levels could not be attributed to changes in intestinal or plasma dipeptidyl peptidase-4 activity, their beneficial effects on glucose tolerance were abolished when mice were pretreated with the GLP-1 receptor antagonist exendin 9–39. Moreover, chronic n-3 PUFA intake prevented the detrimental effects of cHF feeding on glucose-stimulated insulin secretion in the pancreatic islets. Collectively, our data suggest that n-3 PUFA may modulate postprandial glucose metabolism in obese mice through a GLP-1-based mechanism. The significance of these findings in terms of the effective DHA and EPA ratio of the n-3 PUFA concentrate as well as the effect of n-3 PUFA in humans requires further research.