Hepatocellular carcinoma (HCC) is one of the most malignant and poor prognosis tumors, which was increasingly caused by nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) in western countries. In this study, we aimed to investigate the mechanism and therapeutic prospect of berberine in the treatment of NASH-HCC mice. Combination of STZ injection and high fat and high-cholesterol diet (HFHC) was used to establish NASH-HCC model. The effect of berberine intervention is studied from histology, biochemistry and molecular level. Our results showed that administration of berberine to NASH-HCC mice reduced the incidence of tumors and mitigated NASH. Berberine significantly reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose (GLU), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and total cholesterol (TC). Transcriptome sequencing and bioinformatics analysis identified numberous genes and various pathways may participate in the favorite effect of berberine. Specifically, berberine suppressed the expressions of genes related to lipogenesis, inflammation, fibrosis and angiogenesis. Moreover, our results showed that berberine suppressed phosphorylation of p38MAPK and ERK as well as COX2 expression significantly. This suggested berberine achieved its biological functions mainly by regulating inflammation and angiogenesis genes involving p38MAPK/ERK-COX2 pathways. This study demonstrated the anti-tumor effects of berberine and its possible mechanism, providing a potential drug for treating NASH-HCC.