All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. The liver is the main storage organ of vitamin A, but activation of the retinoic acid receptors (RARs) in mouse liver and in human liver cell lines has also been shown. Although atRA treatment improves mitochondrial function in skeletal muscle in rodents, its role in modulating mitochondrial function in the liver is controversial, and little data are available regarding the human liver. The aim of this study was to determine whether atRA regulates hepatic mitochondrial activity. atRA treatment increased the mRNA and protein expression of multiple components of mitochondrial b-oxidation, tricarboxylic acid (TCA) cycle, and respiratory chain. Additionally, atRA increased mitochondrial biogenesis in human hepatocytes and in HepG2 cells with and without lipid loading based on peroxisome proliferator activated receptor gamma coactivator 1a and 1b and nuclear respiratory factor 1 mRNA and mitochondrial DNA quantification. atRA also increased b-oxidation and ATP production in HepG2 cells and in human hepatocytes. Knockdown studies of RARa, RARb, and PPARd revealed that the enhancement of mitochondrial biogenesis and b-oxidation by atRA requires peroxisome proliferator activated receptor delta. In vivo in mice, atRA treatment increased mitochondrial biogenesis markers after an overnight fast. Inhibition of atRA metabolism by talarozole, a cytochrome P450 (CYP) 26 specific inhibitor, increased the effects of atRA on mitochondrial biogenesis markers in HepG2 cells and in vivo in mice. These studies show that atRA regulates mitochondrial function and lipid metabolism and that increasing atRA concentrations in human liver via CYP26 inhibition may increase mitochondrial biogenesis and fatty acid b-oxidation and provide therapeutic benefit in diseases associated with mitochondrial dysfunction.