The objective of this study was to identify genes regulated by thyroid hormone (T3) and associated with tumor invasion. The gene encoding furin, as previously identified by cDNA microarray, is known to be up-regulated by T3 treatment, and stimulated furin production occurs in thyroidectomized rats after administration of T3. Presently, by using serial deletion of the promoter and EMSAs, the T3 response element on the furin promoter was localized to the -6317/-6302 region. T 3-mediated furin up-regulation was cooperative with TGF-β because T3 induction increased after Smad3/4 addition. Furthermore, the invasiveness of HepG2-thyroid hormone receptor (TR) cells was significantly increased by T3 treatment, perhaps due to furin processing of matrix metalloproteinase-2 and -9. In addition, furin up-regulation either by stable over-expression or T3 and/or TGF-β induction was evident in severe-combined immune-deficient mice inoculated with HepG2-TRα1 cells. The HepG2-furin mice displayed a higher metastasis index and tumor size than HepG2-neo mice. Notably, the increased liver and lung tumor number or size in the hyperthyroid severe-combined immune-deficient mice as well as TGF-β mice was attributed specifically to furin overexpression in the HepG2-TRα1 cells. Furthermore, this study demonstrated that furin overexpression in some types of hepatocellular carcinomas is TR dependent and might play a crucial role in the development of hepatocellular carcinoma. Thus, T3 regulates furin gene expression via a novel mechanism or in cooperation with TGF-β to enhance tumor metastasis in vitro and in vivo.