The thyroid transcription factor Pax-8 could bind with the promoter/enhancer of thyroid-specific genes such as thyroglobulin (Tg), thyroperoxidase (TPO) and sodium iodide symporter (NIS), and regulate the expression of these proteins in thyrocyte. Promoting iodide accumulation in tumor cells by re-expression of Pax-8 provides a possible strategy for radioiodine therapy of tumor. Therefore, we investigated the effect of Pax-8 gene transfer on radioiodine therapy of thyroid carcinoma. The human Pax-8 gene was transfected into the human thyroid carcinoma (K1 and F133) cells by the recombinant adenovirus vector. Although the NIS mRNA was not detected, the expression of mRNA and proteins of Tg and TPO in AdPax-8-infected F133 cells were activated by Pax-8. Iodide uptake in thyroid carcinoma cells was reactivated by Pax-8 (increasing 3.3-fold in K1 cells and 5.7-fold in F133 cells). Moreover, Pax-8 promoted iodide organification and the retention time of iodine in Pax-8-expressing cells apparently prolonged in vitro and in vivo (P<0.05). Pax-8-expressing thyroid carcinoma cells were selectively killed by radioiodine. The AdPax-8-infected tumors in vivo clearly visualized in scanning images at 12 h after administration of radioiodine. These results indicate that Pax-8 can promote iodide uptake, and specifically prolong the retention time of iodide in thyroid cancer in vitro and in vivo by promoting the expression of TPO and Tg proteins. Pax-8 gene transfection may lead to effective radioiodine therapy of tumor.