Carbonic anhydrase IX (CAIX) is a membrane-associated carbonic anhydrase (CA) that is overexpressed in a variety of tumor types and associated with increased metastasis, giving a poor prognosis. Transcriptional regulation of transmembrane protein CAIX is complex. We describe further characterization of the 1.2 kb hCA9 promoter, and the effect of TGF-β on the transcriptional activity and expression of hCAIX in Hep3B cells. Transcriptional activity of different promoter regions of hCA9 promoter showed the presence of negative regulatory region between −300 bp and −500 bp of hCAIX promoter. The −116/+38 region was enough for basal transcriptional activity in Hep3B cells. TGF-β upregulates all promoter regions of hCA9 with the highest beig for −466/+38 that has a negatively regulated region. The transcriptional activation of hCA9 promoter by TGF-β is consistent with hCAIX mRNA levels revealed by RT-PCR and hCAIX protein expression levels by flow cytometry in Hep3B cells.