Purpose: The aim of this study was to evaluate the effect of resveratrol on de novo lipogenesis in HepG2 cells caused by high glucose concentrations. Increased lipogenesis in the liver is the main reason for the development of nonalcoholic fatty liver disease (NAFLD) - currently one of the most common chronic liver diseases. In developed countries, this disease is mostly associated with nutritional disorders, resulting from the increasing consumption of monosaccharides. Resveratrol is a natural polyphenol with a promising potential for NAFLD treatment. Methods: The steatosis of HepG2 cells was visualized using the intracellular lipid staining by Nile Red dye with a fluorescence microscope. This study also evaluated the effect of resveratrol on the mitochondrial activity (MitoTracker Green staining), dsDNA (Hoechst 33342 staining) and the viability of HepG2 cells treated with high glucose concentrations (25 and 33 mM). Results: Current study showed that high glucose concentrations induced fat-overloading in HepG2 cells (microvacuolar steatosis occurred in most of the cells). Resveratrol (20 μM) limits the steatosis induction in HepG2 cells by glucose and increased the mitochondrial activity of cells. Resveratrol did not affect the viability of HepG2 cells. Conclusion: This beneficial effect could be helpful in the treatment of NAFLD.