Fibroblast growth factor 21 (FGF21) is a recently discovered metabolic regulator. Interestingly, FGF21 is also known to inhibit Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling from the GH receptor in the liver, where FGF21 mRNA is predominantly expressed. In this study, we tested the hypothesis that FGF21 gene expression in the liver is controlled by GH through STAT5. We found that GH injection to cattle increased FGF21 mRNA expression in the liver. Mapped by a 5'-rapid amplification of cDNA ends assay, transcription of the FGF21 gene in the bovine liver was mainly initiated from a nucleotide 24 bp downstream of a TATA box. The bovine FGF21 promoter contains three putative STAT5-binding sites. EMSA confirmed the ability of them to bind to liver STAT5 protein from GH-injected cattle. Chromatin immunoprecipitation assays demonstrated that GH administration increased the binding of STAT5 to the FGF21 promoter in the liver. Cotransfection analyses showed that GH induced reporter gene expression from the FGF21 promoter in a STAT5-dependent manner. GH also stimulated FGF21 mRNA expression in cultured mouse hepatocytes. These data together indicate that GH directly stimulates FGF21 gene transcription in the liver, at least in part, through STAT5. This finding, together with the fact that FGF21 inhibits GH-induced JAK2-STAT5 signaling in the liver, suggests a novel negative feedback loop that prevents excessive JAK2-STAT5 signaling from the GH receptor in the liver. (Endocrinology 153: 750-758, 2012)