In healthy individuals, peripheral insulin resistance evoked by dietary saturated lipid can be accompanied by increased insulin secretion such that glucose tolerance is maintained. Substitution of long-chain -3 fatty acids for a small percentage of dietary saturated fat prevents insulin resistance in response to high-saturated fat feeding. We substituted a small amount (7%) of dietary lipid with long-chain -3 fatty acids during 4 weeks of high-saturated-fat feeding to investigate the relationship between amelioration of insulin resistance and glucose-stimulated insulin secretion (GSIS). We demonstrate that, despite dietary delivery of saturated fat throughout, this manipulation prevents high-saturated-fat-feeding induced insulin resistance with respect to peripheral glucose disposal and reverses insulin hypersecretion in response to glucose in vivo. Effects of long-chain -3 fatty acid enrichment to lower GSIS were also observed in perifused islets suggesting a direct effect on islet function. However, long-chain -3 fatty acid enrichment led to hepatic insulin resistance with respect to suppression of glucose output and impaired glucose tolerance in vivo. Our data demonstrate that the insulin response to glucose is suppressed to a greater extent than whole-body insulin sensitivity is enhanced by enrichment of a high-saturated fat diet by long-chain -3 fatty acids. Additionally, reduced GSIS despite glucose intolerance suggests that either long-chain -3 fatty acids directly impair the -cell response to saturated fat such that insulin secretion cannot be augmented to normalize glucose tolerance or -cell compensatory hypersecretion represents a response to insulin resistance at the level of peripheral glucose disposal, but not endogenous glucose production.