We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 2–7) and lower (e.g. BHP 14–9) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5′-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 2–7 and BHP 14–9 cells. In BHP 2–7 cells, promoter activity was dependent on an upstream sequence. In BHP 14–9 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1β (HNF-1β) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1β was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1β-positive BHP 2–7 cells, but not in the HNF-1β-negative BHP 14–9 cells. HNF-1β bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1β-specific antibody. Cotransfection of a HNF-1β expression plasmid increased NNMT promoter activity significantly in both HNF-1β-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1β in BHP 14–9 cells increased endogenous NNMT protein levels. In summary, HNF-1β functions as a transcription activator for NNMT gene expression in some papillary thyroid cancer cells.