The “lipotoxicity” hypothesis holds that fat-induced hepatic insulin resistance (FIHIR) may play a major role in the pathogenesis of type 2 diabetes. Berberine has been reported to have antidiabetic properties. However, the molecular mechanisms for this action are not fully clarified. Therefore, we will investigate the gene expression alterations involved in the therapeutic effect of berberine on FIHIR in diabetic hamsters and possible mechanisms. In this study, type 2 diabetic hamsters were induced by high-fat diet with streptozotocin injection. After 9 weeks of berberine-treatment, the gene expression alterations involved in the therapeutic molecular mechanisms of berberine on FIHIR will be studied by microarray technology and real time RT-PCR. Our study demonstrates berberine significantly improved fat-induced insulin resistance and diabetic phenotype in type 2 diabetic hamsters. The alterations of certain metabolism related genes and their main regulators: Liver X receptor (LXR) α, Peroxisome proliferator-activated receptor (PPAR) α and Sterol regulatory element-binding protein (SREBPs) are observed in the liver of treated and untreated diabetic hamsters. Compared with diabetic hamsters, the increased mRNA levels of LXRα and PPARα and the decreased mRNA levels of SREBPs are observed in berberine-treated diabetic hamster. The statistical significance of the expression of hepatic LXRα, SREBPs and PPARα and their certain target genes is found between treated and untreated diabetic hamsters. These results suggest that altered hepatic SREBPs, LXRα and PPARα transcriptional programs possibly involve in the therapeutic mechanisms of berberine on FIHIR in type 2 diabetic hamsters.