Aims: To investigate the relationship between serum phospholipid omega-3 polyunsaturated fatty acids (omega-3 PUFAs) and insulin resistance (IR) in patients with type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). Methods: 51 patients with T2DM and NAFLD (T2DM + NAFLD group), 50 with T2DM alone (T2DM group), 45 with NAFLD alone (NAFLD group), and 42 healthy control subjects (NC group) were studied. Serum omega-3 PUFA profiles were analyzed by gas chromatography, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), and serum lipid concentrations were measured. Insulin resistance was assessed by the homeostasis model assessment method (HOMA-IR). Results: HOMA-IR levels were higher in the T2DM + NAFLD group than in the T2DM, NAFLD and NC groups (p < 0.05), as were ALT, AST, GGT, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) concentrations (p <0.05). Conversely, serum omega-3 PUFA levels were significantly lower in the T2DM + NAFLD group than in the other groups (p < 0.05). The omega-3 PUFA level was negatively correlated with HOMA-IR, TC, LDL-C and TG. Conclusions: Serum phospholipid omega-3 PUFA levels were significantly decreased in patients with T2DM and NAFLD, and were negatively related with insulin resistance. Thus, reduced omega-3 PUFAs may play an important role in the development of T2DM and NAFLD. (c) 2014 Elsevier Inc. All rights reserved.