Although the regulation of thyroid stimulating hormone β-subunit gene (TSHβ) has been intensively studied, the functions of transcription factors involved are not fully understood. The authors found that the -615/-516 promoter region of the TSHβ interacts specifically with nuclear proteins derived from pituitary tissue or from cultured thyrotroph cells. The actual binding site at the nucleotide level, as revealed by DNase I protection assay, includes the consensus sequence for nuclear factor I (NFI). RT-PCR analysis indicated that NFI-B expression is restricted to thyrotroph cells in the anterior pituitary. EMSA and ChIP analysis showed that NFI-B binds most efficiently to the -588/-560 region of TSHβ promoter. The forced expressions of NFI-B markedly reduced TSHβ promoter activity and its mRNA expression. Furthermore, it was also shown that the -588/-560 region is involved in the insulin-mediated repression of the TSHβ. It was of particular interest to observe that NFI-B was recruited to the -588/-560 region of the TSHβ promoter in an insulin-dependent manner. Taken together, this study provides new insights of the delicate regulations of energy metabolism and hormonal homeostasis.