We previously identified Ku proteins and interleukin enhancer binding factor 3 (ILF3) as cofactors for the nuclear receptor farnesoid X receptor and liver receptor homolog-1, respectively. Here we provide further evidence that these cofactors modulate the promoter activity of the nuclear receptor thyroid hormone receptor (TR) target gene, thyroid-stimulating hormone alpha (TSHα), which is negatively regulated by the TR ligand triiodothyronine (T(3)). Ku proteins suppressed TSHα promoter activity independent of T(3), whereas ILF3 enhanced TSHα activity, especially in the presence of T(3). Taken together, our results suggest that Ku proteins and ILF3 function as co-regulators for TR-mediated TSHα expression.