Type 3 deiodinase (DIO3, D3) is reactivated in human neoplasias. Increased D3 levels in papillary thyroid carcinoma (PTC) have been associated with tumor size and metastatic disease. The objective of this study is to investigate the signaling pathways involved inDIO3upregulation in PTC. Experiments were performed in human PTC cell lines (K1 and TPC-1 cells) or tumor samples.DIO3mRNA and activity were evaluated by real-time PCR and ion-exchange column chromatography respectively. Western blot analysis was used to determine the levels of D3 protein.DIO3gene silencing was performed via siRNA transfection.DIO3mRNA levels and activity were readily detected in K1 (BRAFV600E) and, at lower levels, in TPC-1 (RET/PTC1) cells (P<0.007 andP=0.02 respectively). Similarly,DIO3mRNA levels were higher in PTC samples harboring theBRAFV600Emutation as compared with those with RET/PTC1 rearrangement or negative for these mutations (P<0.001). Specific inhibition ofBRAFoncogene (PLX4032, 3 μM), MEK (U0126, 10–20 μM) or p38 (SB203580, 10–20 μM) signaling was associated with decreases inDIO3expression in K1 and TPC-1 cells. Additionally, the blockage of the sonic hedgehog (SHH) pathway by cyclopamine (10 μM) resulted in markedly decreases inDIO3mRNA levels. Interestingly, siRNA-mediatedDIO3silencing induced decreases on cyclin D1 expression and partial G1 phase cell cycle arrest, thereby downregulating cell proliferation. In conclusion, sustained activation of the MAPK and SHH pathways modulate the levels ofDIO3expression in PTC. Importantly,DIO3silencing was associated with decreases in cell proliferation, thus suggesting a D3 role in tumor growth and aggressiveness.