Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that regulates the expression of genes associated with lipid metabolism. Recent studies have suggested that the expression of PPARalpha-dependent fibroblast growth factor 21 (FGF21) plays important roles in adaptation to fasting, such as lipolysis and ketogenesis. We found that a nighttime injection of bezafibrate, a ligand of PPARalpha, effectively induced FGF21 expression, whereas a daytime injection did not affect it. Furthermore, bezafibrate-induced circadian FGF21 expression was abolished in PPARalpha-deficient mice. These observations suggest that bezafibrate-induced circadian FGF21 expression is due to circadian variations in the responsiveness of the PPARalpha system in the liver.