Hepatic lipid metabolic disorders and oxidative stress are involved in the development of non-alcoholic fatty liver disease (NAFLD). This study is to determine the protective effects of Lactobacillus plantarum NA136 on high-fat diet and fructose (HFD/F)-induced NAFLD and to elucidate its underlying molecular mechanisms. Male C57BL/6J mice had been fed with normal diet (ND), HFD/F, or HFD/F supplemented with L. plantarum NA136 for 16 weeks. Treatment with L. plantarum NA136 significantly lowered the body weight gain and decreased the mass of fat tissues, lipids, AST, and ALT levels of HFD/F-treated mice. Our results showed that L. plantarum NA136 activated AMPK pathway to phosphorylate ACC and to suppress the SREBP-1/FAS signaling to inhibit the de novo lipogenesis and increase the fatty acid oxidation. Furthermore, with treatment of L. plantarum NA136, the nuclear translocation of NF-E2-related factor 2 (Nrf2) was also increased which could activate antioxidant pathway. These findings suggested that L. plantarum NA136 improved NAFLD by regulating the fatty acid metabolism and defending against oxidative stress through AMPK and Nrf2 pathways, respectively.