Whether vitamin A (VA) has a role in the development of metabolic abnormalities associated with intake of a high-fat diet (HFD) is unclear. Sprague-Dawley rats after weaning were fed an isocaloric VA sufficient HFD (VAS-HFD) or a VA deficient HFD (VAD-HFD) for 8 weeks. Body mass, food intake, liver and adipose tissue mass, and the hepatic expression levels of key proteins for metabolism were determined. VAD-HFD rats had lower body, liver, and epididymal fat mass than VAS-HFD rats. VAD-HFD rats had lower hepatic protein expression levels of cytochrome P450 26A1, glucokinase, and phosphoenolpyruvate carboxykinase than VAS-HFD rats. VAD-HFD rats had higher protein levels of glycogen synthase kinase (GSK)-3α and lower levels of GSK-3β, but not glycogen synthase, than VAS-HFD rats. VAD-HFD rats had higher hepatic levels of insulin receptor substrate-1 (IRS-1), insulin receptor β-subunit, mitogen-activated protein kinase proteins, and peroxisome proliferator-activated receptor-gamma coactivator 1α mRNA, and lower level of IRS-2 protein than VAS-HFD rats. These results indicate that in a HFD setting, VA deficiency attenuated HFD-induced obesity, and VA status altered the expression levels of proteins required for glucose metabolism and insulin signaling. We conclude that VA status contributes to the regulation of hepatic glucose and lipid metabolism in a HFD setting, and may regulate hepatic carbohydrate metabolism.