Patients with differentiated thyroid cancer (DTC) usually have good prognosis, while those with advanced disease have poor clinical outcomes. This study aimed to investigate the antitumor effects of combination therapy with lenvatinib and I (CTLI) using three different types of DTC cell lines with different profiling of sodium iodide symporter (NIS) status. The radioiodine accumulation study revealed a significantly increased radioiodine uptake in K1-NIS cells after lenvatinib treatment, while there was almost no uptake in K1 and FTC-133 cells. However, lenvatinib administration before radioiodine treatment decreased radioiodine uptake of K1-NIS xenograft tumor in the in vivo imaging study. CTLI synergistically inhibited colony formation and DTC cell migration, especially in K1-NIS cells. Finally, I treatment followed by lenvatinib administration significantly inhibited tumor growth of the NIS-expressing thyroid cancer xenograft model. These results provide important clinical implications for the combined therapy that lenvatinib should be administered after I treatment to maximize the treatment efficacy. Our synergistic treatment effects by CTLI suggested its effectiveness for RAI-avid thyroid cancer, which retains NIS function. This potential combination therapy suggests a powerful and tolerable new therapeutic strategy for advanced thyroid cancer.