Liver X receptor (LXR)-α and -β play a major role in lipid and glucose homeostasis. Their expression and function in the heart is not well characterized. Our aim was to describe the expression of LXRs in the murine heart, and to determine effects of cardiac LXR activation on target gene expression, lipid homeostasis and ischemia. Both LXRα and -β were expressed in heart tissues, HL-1 cells and isolated cardiomyocytes as determined by qRT-PCR. Elevated cardiac expression of LXR target genes and LXRβ was observed 24 h after in vivo permanent coronary artery ligation. The synthetic LXR agonist GW3965 induced mRNA expression of the LXR target genes in HL-1 cells and isolated cardiomyocytes. This was associated with a buildup of intracellular triglycerides and expanding lipid droplets as quantified by confocal microscopy. Mice injected with GW3965 had cardiac LXR activation as judged by increased target gene expression and lipid droplet accumulation. GW3965 in vivo and in vitro increased expression of genes inducing triglyceride synthesis, and altered expression of lipid droplet-binding protein genes. GW3965 protected HL-1 cells against hypoxia-reoxygenation induced apoptosis. LXR activation by GW3965 in vivo prior to heart isolation and perfusion with induced global ischemia and reperfusion improved left ventricular contractile function and decreased infarct size. In conclusion, LXRs are expressed in the murine heart in the basal state, and are activated by myocardial infarction. Activation of LXR by the synthetic agonist GW3965 is associated with intracardiac accumulation of lipid droplets and protection against myocardial ischemia-reperfusion injury.