Aberrantly expressed miRNAs play a crucial role in the development of multiple cancer types， including chordoma. However， the detailed molecular mechanisms are unclear and need to be elucidated. In this study， miRNAs were screened by miRNA array analysis and then confirmed by real-time PCR analysis. We found that miR-16-5p was significantly downregulated in chordoma， and overexpression of miR-16-5p suppressed chordoma cell proliferation， invasion and migration in vitro and in vivo and correlated with the upregulated expression of E-cadherin and downregulated expression of N-cadherin and vimentin. Furthermore， Smad3 was identified as a target of miR-16-5p， and Smad3 was highly expressed in chordoma tissues. Further research showed that knockdown of Smad3 had an effect similar to that of overexpression of miR-16-5p in chordoma cells. Our findings demonstrate that miR-16-5p plays a tumor suppressor role in chordoma progression by targeting Smad3， which could provide a promising prognostic and therapeutic strategy for chordoma treatment.