To give new insight to alterations of cardiac lipid metabolism accompanied by a fructose-rich diet (FRD), rats of both sexes were exposed to 10 % fructose in drinking water during 9 weeks. The protein level and subcellular localization of the main regulators of cardiac lipid metabolism, such as lipin 1, peroxisome proliferator-activated receptor α (PPARα), peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α), carnitine palmitoyltransferase I (CPTI), and CD36 were studied. Caloric intake in fructose-fed rats (FFR) of both sexes was increased. Circulating triacylglyceroles (TAG) and non-esterified fatty acids were increased in male FFR, while females increased visceral adiposity and blood TAG. Total expression of lipin 1 in cardiac cell lysate and its cytosolic and microsomal level were increased in the hearts of male FFR. PPARα and PGC-1α content were decreased in the nuclear extract. In addition, cardiac deposition of TAG in male FFR was elevated, as well as inhibitory phosphorylation of insulin receptor substrate 1 (IRS-1). In contrast, in female FFR, lipin 1 level was increased in nuclear extract only, while overall CPTI expression and phosphorylation of IRS-1 at serine 307 were decreased. The results of our study suggest that fructose diet causes gender-dependent alterations in cardiac lipid metabolism. Potentially detrimental effects of FRD seem to be limited to male rats. Most of the observed changes might be a consequence of elevated expression and altered localization of lipin 1. Increased inhibitory phosphorylation of IRS-1 is possible link between cardiac lipid metabolism and insulin resistance in FFR.