Anoctamin/TMEM16 family members have recently been identified as novel calcium-activated chloride channels, and dysregulation of many family members participates in tumorigenesis and progression. However, the exact role of anoctamin5 (ANO5), one member of this family, in thyroid cancer is still not clarified. In this study, we firstly found that the expression levels of ANO5 was significantly downregulated in thyroid cancer compared to adjacent normal tissue by mining the public GEO database. Subsequently, we further demonstrated that the expression levels of ANO5 was significantly downregulated in 69.5% (57/82) clinical thyroid cancer tissues using real-time PCR assay. Moreover, western blot assay also showed that ANO5 was downregulated in papillary thyroid cancer and follicular thyroid cancer compared to adjacent noncancerous tissues. Furthermore, some biological and functional in vitro experiments proved that ANO5 knockdown promotes thyroid cancer cell migration and invasion but overexpression of ANO5 inhibits these phenotypes. By analyzing gene set enrichment, we found that lower ANO5 expression was positively associated with JAK/STAT3 signaling pathway. Collectively downregulation of ANO5 promotes thyroid cancer cell migration and invasion by affecting JAK/STAT3 pathway.