Increasing reports suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for human cancers, including osteosarcoma. Previous studies have shown that miR-32 was dysregulated in breast and endometrial cancer. However, its biological roles in osteosarcoma remain unclear. In the current study, we found that miR-32 was significantly down-regulated in osteosarcoma tissues, compared with the adjacent normal tissues. In vitro studies further demonstrated that miR-32 mimics were able to suppress, while its antisense oligos promoted cell proliferation in Saos-2 and U2OS cells. At the molecular level, our data further revealed that expression of Sox9 was negatively regulated by miR-32. Therefore, our results identify an important role for miR-32 in the osteosarcoma through regulating Sox9 expression.