Nonalcoholic fatty liver disease (NAFLD) is a hepatic ailment with a rapidly increasing incidence due to dietary hyper nutrition and subsequent obesity. Discovering effective natural materials and herbs can provide alternative and complementary medical treatments to current chemical pharmaceuticals. To develop an effective natural agent for NAFLD, we formulated a combination of 10 herb mixtures and observed lipid-lowering efficacy and to investigate the preventive effects of a compound of Chinese Herbal Formula (CHF03) on a high-fat diet (HFD) induced model of NAFLD in mice in vitroand in vivo. The CHF03 groups were fed with HFD and orally administered 10 g/kg CHF03 once a day orally. HE staining was performed to analyze pathologic changes of the liver; a Transmission Electron Microscopy assay was performed to measure the ultrastructural alterations of the mitochondria, and Western blotting was performed to detect the expression of gene proteins related to lipid metabolism and in?ammation. To further examine the safety of CHF03, the composition of CHF03 was analyzed by liquid chromatography-mass spectrometry (LC/MS) and acute toxicity and maximum tolerable dose was performed in mice. The results of histomorphological and ultra-structural changes showed that CHF03 could effectively inhibit the occurrence of non-alcoholic fatty liver induced by high fat. CHF03 attenuated high-fat diet-induced oxidative stress, which was confirmed by measuring the level of oxidative stress markers (GSH, GSH-Px, MDA, SOD and CAT). The preventive effects of CHF03 against lipogenesis and inflammatory were mediated by the inhibition of protein acetyl-CoA carboxylase (ACC1), fatty acid synthase (FAS) and nuclear factor κB (NFκB). Moreover, CHF03 dose-dependently inhibited lipid accumulation and gene expressions involved in lipogenesis and related regulators (SREBP-1c, CPT1, FAS, ACC1, ApoA and NFκB) and prevented the PA-induced lipid accumulation in hepatocytes. Additionally, CHF03 inhibited the PA-induced increase in the expression, nuclear localization, and transcriptional activities of NFκB. These results suggest that CHF03 to suppress HFD-induced NAFLD in part through the activation of NF-κB.