MicroRNAs (miRNAs) are emerging as critical regulators in carcinogenesis and tumor progression. Recently,miR-99a has been reported as a tumor suppressor gene in various human cancers, but its functions in the contextof anaplastic thyroid cancer (ATC) remain unknown. In this study, we reported that miR-99a was commonlydownregulated in ATC tissue specimens and cell lines with important functional consequences. Overexpressionof miR-99a not only dramatically reduced ATC cell viability by inducing cell apoptosis and accumulation of cellsat G1 phase, but also inhibited tumorigenicity in vivo. We then screened and identified a novel miR-99a target,mammalian target of rapamycin (mTOR), and it was further confirmed by luciferase assay. Up-regulation ofmiR-99a would markedly reduce the expression of mTOR and its downstream phosphorylated proteins (p-4EBP1and p-S6K1). Similar to restoring miR-99a expression, mTOR down-regulation suppressed cell viabilityand increased cell apoptosis, whereas restoration of mTOR expression significantly reversed the miR-99aantitumor activity and the inhibition of mTOR/p-4E-BP1/p-S6K1 signal pathway profile. In clinical specimensand cell lines, mTOR was commonly overexpressed and its protein levels were statistically inversely correlatedwith miR-99a expression. Taken together, our results demonstrated for the first time that miR-99a functions asa tumor suppressor and plays an important role in inhibiting the tumorigenesis through targeting the mTOR/p-4E-BP1/p-S6K1 pathway in ATC cells. Given these, miR-99a may serve as a novel prognostic/diagnostic andtherapeutic target for treating ATC.