Fibrates are widely used as lipid lowering drugs acting as peroxisome proliferator-activated receptors α (PPARα) agonists and modulating the expression of several genes involved in lipid and lipoprotein metabolism. Much less is known on the effect of fibrates in HDL structure and composition. Therefore, we examined whether fenofibrate induces quantitative and/or qualitative modifications in HDL metabolism in the rabbit, an animal that, contrary to rodents and similar to humans, is less sensitive to peroxisome proliferators. We first demonstrated that 3-week treatment with fenofibrate (250 mg/kg/day) induced an important increase in serum apolipoprotein A-I, HDL-cholesterol and HDL-phospholipids concentrations and a relative enrichment in HDL cholesteryl ester content. Moreover, the fatty acid profiles from fenofibrate-treated rabbits displayed a dramatic increase in the serum or HDL C18:3 ω6 to C18:2 ω6 ratio suggesting higher Δ6 desaturase activity. In addition, HDL from fenofibrate-treated animals exhibited higher relative proportions of sphingomyelin, phosphatidylinositol and phosphatidylethanolamine. We then reported that fenofibrate induced major changes in the physical characteristics of HDL, mainly a higher size and a faster mobility on agarose gel electrophoresis. Finally, serum or HDL from treated rabbits exhibited higher capacity to promote cholesterol efflux from Scavenger receptor class B type I (SR-BI)-rich Fu5AH cells compared to controls. Our findings demonstrate that fenofibrate has beneficial effects in rabbits by increasing the mass of the circulating HDL pool and by modifying their composition transforming them as better acceptors of cellular cholesterol through SR-BI pathway. These effects of fenofibrate might contribute to its benefits on the prevention and treatment of atherosclerosis.