Background: In thyroid cancers, a reduction in the expression of the sodium/iodide symporter (NIS) is observed concomitant with a diminution in cancer cell differentiation. The beta-catenin/LEF1 pathway emerges as a crucial regulatory pathway influencing the functional expression of NIS in human thyroid cancer cells. Further research is required to comprehensively elucidate the role of NIS overexpression in impeding the progression of thyroid cancer cells. Methods: Human papillary thyroid carcinoma (PTC) cell lines, specifically PTC-1 and KTC-1, were subjected to Scratch and Transwell assays, colony formation, and tumor sphere formation tests to investigate invasion and migration, focusing on the impact of NIS overexpression. The assessment involved the use of western blot to analyze the expression levels of beta-catenin, NIS, CD133, SRYrelated HMG box2 (Sox2), lymphoid enhancer-binding factor 1 (LEF-1), NANOG, octamerbinding transcription factor 4 (Oct4), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), and epithelial cellular adhesion molecule (EpCAM). Statistical analysis was conducted using SPSS version 20.0, and the graphs were developed using GraphPad Prism 7 (GraphPad Software, Inc.). Results: Our observations revealed that Nthy-ori-3-1 cell lines exhibited notably higher average expression levels of NIS, yet significantly lower levels of LEF-1 and beta-catenin compared to PTC-1 and KTC-1 cell lines. Furthermore, the overexpression of beta-catenin resulted in reduced binding of LEF-1 to NIF promotion but concurrently increased the expression of NIS. The downregulation of NIS markedly enhanced the expression of ALDH1A1, CD133, OCT4, Nanog, SOX2, and EpCam-all of which are targets within the Wnt/beta-catenin signaling pathway. Conversely, the upregulation of NIS suppressed the expression of these proteins. Moreover, cells treated with beta-catenin activators demonstrated an increased capability to form more spheroids and displayed heightened aggressiveness. Conversely, the NIS overexpression (OE) group exhibited suppressed abilities in invasion and colony formation. Conclusion: Thyroid cancer cells exhibit diminished expression of NIS, and the invasion and maintenance of stem cells in thyroid cancer cells were hindered by NIS OE through the inhibition of the beta-catenin/LEF-1 pathway. Further research is warranted to comprehensively assess this outcome, which holds promise as a potential targeted treatment for thyroid cancer.