Alterations in cardiac glucose and fatty acid metabolism are possible contributors to the pathogenesis of heart failure in obesity. Here we examined the effect of leptin, the product of the obese (ob) gene, on metabolism in murine cardiomyocytes. Neither short-term (1 hour) nor long-term (24 hours) treatment with leptin (60 nmol/L) altered basal or insulin-stimulated glucose uptake and oxidation, glycogen synthesis, insulin receptor substrate 1 tyrosine, Akt, or glycogen synthase kinase 3beta phosphorylation. Extracellular lactate levels were also unaffected by leptin. However, leptin increased basal and insulin-stimulated palmitate uptake at both short and long exposure times and this corresponded with increased cell surface CD36 levels and elevated fatty acid transport protein 1 (FATP1) and CD36 protein content. Whereas short-term leptin treatment increased fatty acid oxidation, there was a decrease in oxidation after 24 hours. The former corresponded with increased acetyl coenzyme A carboxylase phosphorylation and the latter with increased expression of this enzyme. The discrepancy between uptake and oxidation of fatty acids led to a transient decrease in intracellular lipid content with lipid accumulation ensuing after 24 hours. In summary, we demonstrate that leptin did not alter glucose uptake or metabolism in murine cardiomyocytes. However, fatty acid uptake increased while oxidation decreased over time leading to intracellular lipid accumulation, which may lead to lipotoxic damage in heart failure.