Glycine N-methyltransferase (GNMT) is a tumor susceptibility gene for both hepatocellular carcinoma and prostate cancer. We have previously characterized GNMT genomic structure and mapped its chromosomal localization to 6p12. For this study we identified a GNMT transcriptional start site at the 14th position upstream of the ATG codon. Electrophoretic mobility shift assay results indicate binding of the nuclear factor-Y (NF-Y) transcription factor to the CCAAT box (-71/-67) of the GNMT gene. Mutation assay results suggest that the nucleotide sequence in the -56/-47 region is a binding site for a putative transcriptional factor. The TATA-less core promoter (-133/+14) contains three major elements: an Sp1 site, CCAAT box, and a novel box within the CTGTCGGCTG sequence. One functional xenobiotic response element (XRE) located at the -104/-82 region is inducable by benzo[a]pyrene treatment. We believe our results have value for the study of GNMT transcriptional regulation.