Previous studies have shown that starvation or consumption of a high fat, low carbohydrate (HF-LC) ketogenic diet induces hepatic fibroblast growth factor 21 (FGF21) gene expression in part by activating the peroxisome proliferator-activated receptor-α (PPARα). Using primary hepatocyte cultures to screen for endogenous signals that mediate the nutritional regulation of FGF21 expression, we identified two sources of PPARα activators (i.e. nonesterified unsaturated fatty acids and chylomicron remnants) that induced FGF21 gene expression. In addition, we discovered that natural (i.e. bile acids) and synthetic (i.e. GW4064) activators of the farnesoid X receptor (FXR) increased FGF21 gene expression and secretion. The effects of bile acids were additive with the effects of nonesterified unsaturated fatty acids in regulating FGF21 expression. FXR activation of FGF21 gene transcription was mediated by an FXR/retinoid X receptor binding site in the 5'-flanking region of the FGF21 gene. FGF19, a gut hormone whose expression and secretion is induced by intestinal bile acids, also increased hepatic FGF21 secretion. Deletion of FXR in mice suppressed the ability of an HF-LC ketogenic diet to induce hepatic FGF21 gene expression. The results of this study identify FXR as a new signaling pathway activating FGF21 expression and provide evidence that FXR activators work in combination with PPARα activators to mediate the stimulatory effect of an HF-LC ketogenic diet on FGF21 expression. We propose that the enhanced enterohepatic flux of bile acids during HF-LC consumption leads to activation of hepatic FXR and FGF19 signaling activity and an increase in FGF21 gene expression and secretion.