Loss of liver fatty acid binding protein (L-FABP) decreases long chain fatty acid uptake and oxidation in primary hepatocytes and in vivo. On this basis, L-FABP gene ablation would potentiate high-fat diet-induced weight gain and weight gain/energy intake. While this was indeed the case when L-FABP null (-/-) mice on the C57BL/6NCr background were pair-fed a high-fat diet, whether this would also be observed under high-fat diet fed ad libitum was not known. Therefore, this possibility was examined in female L-FABP (-/-) mice on the same background. L-FABP (-/-) mice consumed equal amounts of defined high-fat or isocaloric control diets fed ad libitum. However, on the ad libitum-fed high-fat diet the L-FABP (-/-) mice exhibited: (1) decreased hepatic long chain fatty acid (LCFA) β-oxidation as indicated by lower serum β-hydroxybutyrate level; (2) decreased hepatic protein levels of key enzymes mitochondrial (rate limiting carnitine palmitoyl acyltransferase A1, CPT1A; HMG-CoA synthase) and peroxisomal (acyl CoA oxidase 1, ACOX1) LCFA β-oxidation; (3) increased fat tissue mass (FTM) and FTM/energy intake to the greatest extent; and (4) exacerbated body weight gain, weight gain/energy intake, liver weight, and liver weight/body weight to the greatest extent. Taken together, these findings showed that L-FABP gene-ablation exacerbated diet-induced weight gain and fat tissue mass gain in mice fed high-fat diet ad libitum--consistent with the known biochemistry and cell biology of L-FABP.