NIS is a potent iodide transporter encoded by the gene. Its expression is reduced in papillary thyroid carcinoma (PTC). In this study we analyzed the impact of miR-181a-5p on NIS expression in the context of PTC. We used real-time PCR to analyze the expression of and miR-181a-5p in 49 PTC/normal tissue pairs. Luciferase assays and mutagenesis were performed to confirm direct binding of miR-181a-5p to the 3'UTR of and identify the binding site. The impact of modulation of miR-181a-5p using appropriate plasmids on endogenous NIS and radioactive iodine accumulation was verified. We confirmed downregulation of and concomitant upregulation of miR-181a-5p in PTC. Broadly used algorithms did not predict the binding site of miR-181a-5p in 3'UTR of , but we identified and confirmed the binding site through mutagenesis using luciferase assays. In MCF7 and HEK293-flhNIS cell lines, transfection with mir-181a-expressing plasmid decreased endogenous , whereas silencing of miR-181a-5p increased it. We observed similar tendencies in protein expression and radioactive iodine accumulation. This study shows for the first time that miR-181a-5p directly regulates expression in the context of PTC and may decrease efficacy of radioiodine treatment. Accordingly, miR-181a-5p may serve as an emerging target to enhance the efficacy of radioactive iodine therapy.