Deregulated microRNAs and their roles in tumorigenesis are still largely unknown. Here, we focused on the roles of miR-143 in osteosarcoma, as previous reports have suggested its importance in some other types of cancer. We found that miR-143 was down-regulated in osteosarcoma cell lines and primary tumor samples, and the restoration of miR-143 reduced cell viability, promoted cell apoptosis and suppressed tumorigenicity. Additionally, Bcl-2, an important antiapoptotic molecule, was identified to be a novel direct target of miR-143, and the proapoptotic function of miR-143 is further suggested to be mainly through the targeting of Bcl-2 expression. Collectively, our data identify the important roles of miR-143 in osteosarcoma pathogenesis and indicate its potential application in cancer therapy.