Liver X receptors (LXRs) are master transcription factors regulating cholesterol and fatty acid metabolism. Treatment of C57B6 mice with a specific synthetic LXR agonist, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317), resulted in elevated high-density lipoprotein (HDL) cholesterol as well as plasma and liver triglycerides. Peroxisome proliferator-activated receptor-α (PPARα) agonists are known to induce peroxisomal fatty acid β-oxidation and also mediate HDL cholesterol metabolism. We have explored the hypothesis that simultaneous activation of PPARα and LXR may lead to additive effects on HDL cholesterol elevation as well as attenuation of triglyceride accumulation. Coadministration of T0901317 and the specific PPARα agonist [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy14643)] in mice led to synergistic elevation of HDL cholesterol that was primarily associated with enlarged HDL particles enriched with apoE and apoAI. Liver phospholipid transfer protein (PLTP) mRNA and plasma PLTP activity were additively elevated, suggesting a role of PLTP in the observed HDL cholesterol elevation. Moderate increases in plasma triglyceride levels induced by LXR activation was reduced, whereas the accumulation of triglyceride in the liver was not altered upon coadministration of the PPARα agonist. Peroxisomal fatty acid β-oxidation in the liver was dramatically elevated upon PPARα activation as expected. Interestingly, activation of LXRs via T0901317 also led to a significant increase in peroxisomal fatty acid β-oxidation. Sterol regulatory element binding protein 1c expression was dramatically up-regulated by the LXR agonist but was not changed with PPARα agonist treatment. Liver lipoprotein lipase expression was additively increased upon LXR agonist and PPARα agonist coadministration. Our studies mark the first exploration of nuclear receptor interplay on lipid homeostasis in vivo.