OBJECTIVE It is known that insulin resistance has an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and that serum 25-hidroksivitamin D3 [25-(OH)D] levels are found low in the presence of insulin resistance. Metabolic syndrome (MetS) is characterized by insulin resistance. The purpose of the present study was to determine the levels of 25-(OH)D and the frequency of MetS in patients with NAFLD, and to evaluate the association of 25-(OH)D with the histology of NAFLD and metabolic parameters. METHODS Sixty-three patients with NAFLD confirmed by liver biopsy (29 females and 34 males, mean age 42.70±9.82 years) and 46 healthy controls (16 females and 30 males, mean age 37.54±8.56 years) were included in the study. International Diabetes Federation criteria were used for MetS diagnosis. Insulin resistance was determined according to the Homeostasis Model of Assessment (HOMA-IR) method. The groups were compared for 25-(OH)D levels and MetS frequencies. Correlation analysis was used to evaluate relationships between 25-(OH)D and metabolic parameters and/or NAFLD histology. RESULTS 25-(OH)D levels were lower in the NAFLD group compared to the control group (36.06±13.07 ng/mL vs. 51.19±23.45 ng/mL, respectively, P<0.01), while MetS frequency was higher (66.7% vs. 15.2%, P<0.01). In the NAFLD group, 25-(OH)D levels were negatively correlated with non-alcoholic steatohepatitis scores and HOMA-IR (r=-0.317, P=0.011 and r=-0.437, P=0.001, respectively). CONCLUSIONS The present study demonstrated higher frequency of MetS and lower levels of 25-(OH)D in patients with NAFDL, and a negative association of 25-(OH)D levels with non-alcoholic steatohepatitis scores and insulin resistance.