Gypenosides (GP) are a type of traditional Chinese medicine (TCM) extracted from plants and commonly applied for treatment of metabolic diseases. This study aims to explore the effects of GP extracts on alleviating non-alcoholic fatty liver disease (NAFLD). In this experiment, C57BL/6 J mice were randomly assigned into normal diet control (ND), HFHC (high-fat and high-cholesterol) and HFHC + GP (GP) groups. Mice in HFHC group were fed HFHC diet combined with fructose drinking water for 12 weeks to induce the animal model of NAFLD, followed by ordinary drinking water until the end of the experiment. In the HFHC + GP group, mice were fed HFHC diet combined with fructose drinking water for 12 weeks, followed by GP-containing drinking water till the end. Mouse body weight was measured weekly. After animal procedures, mouse liver and serum samples were collected. It is shown that GP administration reduced body weight, enhanced the sensitivity to insulin resistance (IR) and decreased serum levels of ALT, AST and TG in NAFLD mice. In addition, GP treatment alleviated steatohepatitis, and downregulated ACC1, PPARγ, CD36, APOC3 and MTTP levels in mice fed with HFHC diet. Furthermore, GP treatment markedly improved intestinal microbiota, and reduced relative abundance ratio of Firmicutes / Bacteroidetes in the feces of NAFLD mice. Our results suggested that GP alleviated NAFLD in mice through improving intestinal microbiota.