The aim of this study was to investigate the effects of vitamin D supplementation on serum aminotransferases, insulin resistance, oxidative stress, and inflammatory biomarkers in adult patients with non-alcoholic fatty liver disease (NAFLD). Fifty-three patients with NAFLD were enrolled in a parallel, double-blind, placebo-controlled study. The patients were randomly allocated to receive either one oral pearl consisting of 50,000 IU vitamin D3 (n = 27) or a placebo (n = 26), every 14 days for 4 months. Serum aminotransferases, high-sensitive C-reactive protein (hs-CRP), tumor necrosis factor α, malondialdehyde (MDA), total antioxidant capacity, transforming growth factor β1, as well as grade of hepatic steatosis and homeostasis model assessment of insulin resistance were assessed pre- and post-intervention. In patients who received vitamin D supplement compared to the controls, the median of serum 25(OH)D3 significantly increased (16.2 vs. 1.6 ng/ml, P < 0.001). This increase accompanied by significant decrease in serum MDA (-2.09 vs. -1.23 ng/ml, P = 0.03) and near significant changes in serum hs-CRP (-0.25 vs. 0.22 mg/l, P = 0.06). These between-group differences remained significant even after controlling for baseline covariates. Other variables showed no significant changes. Improved vitamin D status led to amelioration in serum hs-CRP and MDA in patients with NAFLD. This might be considered as an adjunctive therapy to attenuate systemic inflammation and lipid peroxidation alongside other treatments for NAFLD patients.