Fibrates, including fenofibrate, exert their biological effects by binding peroxisome proliferator-activated receptor alpha (PPARalpha), a member of the nuclear receptor superfamily of ligand-activated transcription factors. Treatment with PPARalpha agonists enhances fatty acid oxidation, decreases plasma triglycerides, and may promote reverse cholesterol transport. In addition, fibrate administration can reduce intestinal cholesterol absorption in patients, although the molecular mechanism for this effect is unknown. Because Niemann-Pick C1-Like 1 (NPC1L1) is already known to be a critical protein for cholesterol absorption, we hypothesized that fenofibrate might modulate NPC1L1 expression to alter intestinal cholesterol transport. Here, we find that fenofibrate-treated wild-type mice have decreased fractional cholesterol absorption (35-47% decrease) and increased fecal neutral sterol excretion (51-83% increase), which correspond to decreased expression of NPC1L1 mRNA and protein (38-66% decrease) in the proximal small intestine. These effects of fenofibrate are dependent on PPARalpha, as Ppar alpha-knockout mice fail to respond like wild-type littermates. Fenofibrate affects the ezetimibe-sensitive pathway and retains the ability to decrease cholesterol absorption and NPC1L1 mRNA expression in chow-fed liver X receptor alpha/beta-double-knockout mice and high-cholesterol- or cholic acid-fed wild-type mice. These data demonstrate that fenofibrate specifically acts via PPARalpha to decrease cholesterol absorption at the level of intestinal NPC1L1 expression.