We previously reported that a high-fat and high-cholesterol (HFHC) diet for 12 weeks induced non-alcoholic steatohepatitis (NASH) and influenced major CYP subtype gene expression levels and activities in a mouse model. In the present study, we determined the effects of the HFHC diet on CYP expression levels and activities prior to the establishment of NASH. When male C57BL/6J mice were fed the HFHC or a normal chow diet (Control) ad libitum for 4 weeks, body weights were significantly lower, whereas liver weights and hepatic lipid contents were significantly higher in the HFHC group than in the Control group. Under these conditions, hepatic microsomal luciferin-H (human CYP2C9 substrate) hydroxylation activity was significantly lower in the HFHC group than in the Control group. In order to investigate drug efficacy in mice fed the HFHC diet, an intraperitoneal glucose tolerance test was conducted with or without a pretreatment with tolbutamide (a CYP2C substrate) after 4 weeks of feeding. The plasma glucose-lowering effects of tolbutamide were attenuated in the HFHC group even though luciferin-H hydroxylation activity was suppressed in this group. The reason for this discrepancy was attributed to the mRNA expression levels of Cyp2c44 being lower and those of Cyp2c29 and Cyp2c66, which are involved in the metabolism of tolbutamide, being higher in the HFHC group than in the Control group. These results indicate that the expression of Cyp2c in the liver is influenced by the HFHC diet prior to the establishment of NASH and its regulation differed among the subtypes examined.