Dietary habits and gut microbiota play an essential role in non-alcoholic fatty liver disease (NAFLD) and related factors such as insulin resistance and de novo lipogenesis. In this study, we investigated the protective effects of CBA7346, isolated from the gut of healthy Koreans, on mice with high-fat diet (HFD)-induced NAFLD. Administration of CBA7346 reduced body and liver weight gain, serum alanine aminotransferase and aspartate aminotransferase levels, liver steatosis, and liver triglyceride levels in mice on an HFD; the strain also decreased homeostatic model assessment for insulin resistance values, as well as serum cholesterol, triglyceride, lipopolysaccharide, leptin, and adiponectin levels in mice on an HFD. Moreover, CBA7346 controlled fatty liver disease by attenuating steatosis and inflammation and regulating de novo lipogenesis-related proteins in mice on an HFD. Taken together, these findings suggest that CBA7346 ameliorates HFD-induced NAFLD by reducing insulin resistance and regulating de novo lipogenesis in obese mice.