Thyroid hormone, T3, regulates cell metabolism, differentiation, and development. cDNA microarrays were performed to study the mechanism of target gene regulation after T3 treatment in a thyroid hormone receptor-α (TRα)-overexpressing hepatoma cell line (HepG2-TRα). The differentially expressed target genes are several metabolic enzymes, including dehydroepiandrosterone-sulfotransferase family 1A member 2 (SULT2A1). Enzyme SULT2A1 was elevated roughly 5-fold at the protein level and 9-fold increase at the mRNA level after 48 h T3 treatment in HepG2-TRα cells. Cycloheximide inhibited T3-induced SULT2A1 expression, suggesting that regulation was indirect. SULT2A1 has been reported to be regulated by the two transcription factors, steroidogenic factor 1 (SF1) and GATA, in the human adrenal gland. T3 induced a 2.5- to 3.5-fold elevation of SF1 at the protein level and a 6.2-fold increase at the RNA level in HepG2-TRα cells. About seven SF1 binding sites exist on the SULT2A1 gene. To identify and localize the critical SF1 binding site, series of deletion mutants of SULT2A1 promoter fragments in pGL2 plasmid were constructed. The promoter activity of the SULT2A1 gene was enhanced about 2.8- to 7.1-fold by T3. The −228 SF1 binding site was identified as the most critical site because deleting this region reduced T3-induced expression. Transcription factor SF1 application enhanced the −228 but not −117 reporter plasmid activities. SULT2A1 and SF1 up-regulation at protein and RNA levels in thyroidectomized rats occurred after T3 application. In summary, this work demonstrated that the SULT2A1 gene was mediated by SF1 and indirectly regulated by T3. Further study is required to elucidate the physiological importance of SULT2A1 induction mediated by T3.