MicroRNAs of the miR-16 and miR-34 families have been reported to inhibit cell cycle progression, and their loss has been linked to oncogenic transformation. Utilizing a high-throughput, genome-wide screen for miRNAs and mRNAs that are differentially regulated in osteosarcoma (OS) cell lines, we report that miR-449a and miR-424, belonging to the miR-34 and miR-16 families, respectively, target the major S/G phase cyclin, cyclin A2 (), in a bipartite manner. We found that the 3'-UTR of is recognized by miR-449a, whereas the coding region is targeted by miR-424. Of note, we observed loss of both miR-449a and miR-424 in OS, resulting in derepression of CCNA2 and appearance of aggressive cancer phenotypes. Ectopic expression of miR-449a and miR-424 significantly decreased cyclin A2 levels and inhibited proliferation rate, migratory potential, and colony-forming ability of OS cells. To further probe the roles of miR-449a and miR-424 in OS, we developed an OS mouse model by intraosseous injection of U2OS cells into the tibia bone of NOD- mice, which indicated that miR-449a and miR-424 co-expression suppresses tumor growth. On the basis of this discovery, we analyzed the gene expression of human OS biopsy samples, revealing that miR-449a and miR-424 are both down-regulated, whereas cyclin A2 is significantly up-regulated in these OS samples. In summary, the findings in our study highlight that cyclin A2 repression by miRNAs of the miR-16 and miR-34 families is lost in aggressive OS.