Fish-oil supplementation decreases serum triacylglycerols but may worsen hyperglycemia in patients with non-insulin-dependent diabetes mellitus. The reason for the possible deterioration of glycemia is unclear. We examined whether inhibition of triacylglycerol synthesis by n-3 fatty acids changes lipolysis, glycerol gluconeogenesis, or fatty acid oxidation. Nine obese patients with non-insulin-dependent diabetes mellitus participated in a randomized double-blind crossover study in which 6 wk of n-3 fatty acid supplementation (12 g fish oil) was compared with 6 wk of corn plus olive oil. Serum triacylglycerols decreased by 30% during n-3 fatty acid supplementation. Glycerol gluconeogenesis ([U-14C]glycerol) increased by 32%. However, overall glucose production ([3-3H]glucose), glycemic control, and fatty acid oxidation remained unchanged. Thus, 6 wk of n-3 fatty acid supplementation lowers triacylglycerols in patients with non-insulin-dependent diabetes mellitus without worsening glycemic control. However, n-3 fatty acid supplementation increases glycerol gluconeogenesis, which could contribute to deterioration of glycemic control during long-term treatment with high doses of fish-oil supplements.