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Background: It has been shown that low concentrations of high-density lipoprotein cholesterol (HDL-C) are associated with non-alcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM). HDL2b, a major subfraction of high-density lipoprotein (HDL), is more significantly correlated with coronary heart disease (CHD) compared with total HDL. In this study, we analysed HDL2b in a cohort of Chinese T2DM subjects with or without NAFLD., Methods: A highly sensitive and reliable microfluidic chip method was adopted to measure HDL2b. In total, 48 T2DM patients with NAFLD diagnosed by a B-ultrasound were enrolled from our Beijing Community Pre-Diabetes (BCPD) study cohort. A total of 48 age and gender matched diabetic controls without NAFLD were selected from the same population., Results: Clinical characteristics and serum biochemical analyses demonstrated a significantly increased body mass index (BMI), waist circumference, homeostasis model assessment-insulin resistant index (HOMA-IR), total cholesterol (TC), and triglyceride (TG) concentrations in the NAFLD group. However, the concentrations of HDL2b and its ratio to total HDL in NAFLD patients was decreased, compared with controls (p<0.01). Significantly increased concentrations of high sensitive C-reactive protein (hs-CRP) (p<0.01) were also found. Multifactor logistic regression analysis showed that BMI and TG were the predominant risk factors for fatty liver, while HDL2b was a protective factor., Conclusions: T2DM patients with NAFLD have characteristics including obesity, marked insulin resistance, high TG, high hs-CRP, low HDL2b and a low HDL2b ratio to total HDL. These factors may increase the incidence of atherosclerosis as well as the risk of CHD.