Fenofibrate belongs to the group of hypolipidemic fibrates that act as activators of the peroxisome proliferator-activated receptor-α (PPARα), which is a regulator of bile acid synthesis, metabolism, and transport. The present study aimed at evaluating the effects of fenofibrate on the circulating bile acid profile in humans. A study population of 200 healthy individuals comprising both genders completed a 3-week intervention with fenofibrate, and 17 bile acid species were measured in serum samples drawn before and after fenofibrate treatment. Fenofibrate caused significant reductions in levels of chenodeoxycholic (CDCA) (26.4%), ursodeoxycholic (UDCA) (30.5%), lithocholic (LCA) (18.4%), deoxycholic (DCA) (22.3%), and hyodeoxycholic (HDCA) (19.2%) acids. A gender-related difference was observed in the responses of various bile acids, and the total bile acid concentration was significantly reduced only in men (18.6%), whereas it remained almost unchanged in women (0.36%). This difference suggests that fenofibrate would be more efficient at reducing bile acid toxicity in men than in women in cholestatic liver diseases.