Transcriptional regulation of downstream gene expression by thyroid hormone (T3) is mediated by the thyroid hormone receptor (TR). T3 binding induces a complicated transition, where TR converts from a transcriptional repressor into a transcriptional activator and instigates downstream gene transcription. Binding of T3 to TR also induces the degradation of TR, resulting in desensitization of the cells to further T3 treatment. It has been shown that phosphorylation of TR plays a critical role in its activity and stability after T3 binding. However, the kinases in control of phosphorylating TR in the nucleus have not been identified. In this study we demonstrate that MAPKs are possible candidates responsible for the nuclear phosphorylation of TR. Suppression of MAPKs with specific inhibitors repressed TR transcriptional activity and antagonized okadeic acid-induced TR transcriptional activity potentiation. Overexpression of the MAPK activator, MKK6, and its constitutively active mutant, MKK6EE, significantly increased TR activity and protected TR from degradation. Involvement of the 26S ubiquitin proteasome in hormone binding-induced TR degradation was also examined. We found that MAPKs enhanced the DNA binding affinity of TR. Our results suggest that MAPKs are the major kinases responsible for the nuclear phosphorylation of TR and are critical factors modulating the transcriptional activity and protein stability of TR subsequent to ligand binding.