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 c-DNA microarray, is known to be up-regulated by T₃ treatment, and stimulated furin production occurs in thyroidectomized rats following administration of T₃. Presently, by using serial deletion of the promoter and electrophoretic mobility shift assays, the T₃ response element on the furin promoter was localized to the -6317/-6302 region. T₃-mediated furin up-regulation was cooperative with TGF-, since T₃ induction increased following Smad3/4 addition. Further, the invasiveness of HepG2-TR cells was significantly increased by T₃ treatment, perhaps due to furin processing of matrix metalloproteinase-2 and -9. Additionally, furin up-regulation either by stable over-expression or by T₃ and/or TGF- induction was evident in severe-combined immune-deficient (SCID) mice inoculated with HepG2-TR1 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 hyper-thyroid SCID mice as well as TGF- mice was attributed specifically to furin over-expression in the HepG2-TR1 cells. Further, this study demonstrated that furin over-expression in some types of hepatocellular carcinomas (HCCs) is TR-dependent and might play a crucial role in the development of HCC. Thus, T₃ regulates furin gene expression via a novel mechanism or in cooperation with TGF- to enhance tumor metastasis in vitro and in vivo.