Kruppel-like factor 6 (KLF6) is a tumor suppressor gene frequently downregulated in a number of human cancers, including osteosarcoma. However, the role of KLF6 in osteosarcoma remains unclear. This study was aimed at investigating the effects of KLF6 on osteosarcoma cell biological behavior. First, the expression of KLF6 in osteosarcoma cell lines (MG63, SaOS-2, U2OS, and HOS) and a human osteoblastic cell line (hFOB1.19) was detected by Western blotting. Results showed that KLF6 displayed a significant downregulation in osteosarcoma cell lines (MG63, SaOS-2, U2OS, and HOS) compared with human osteoblastic cell line (hFOB1.19). To investigate the role of KLF6 in osteosarcoma cell proliferation, apoptosis, and invasion, we generated human osteosarcoma MG63 cells in which KLF6 was either overexpressed or depleted. The MG63 cell viability, cycle, apoptosis, and invasive ability were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining, propidium iodide (PI) staining, Annexin-V-FITC/PI double staining, and Transwell invasion experiment, respectively. Results showed that the viability, proliferation, and invasive abilities were suppressed, and the apoptosis was enhanced in MG63 cells with overexpression of KLF6. The viability, proliferation, and invasive abilities were improved, and the apoptosis was inhibited in MG63 cells with knockdown of KLF6. At the same time, these molecules, including p21, bcl-2, and MMP-9, associated with the events about cell cycle, apoptosis, and invasion, were detected. Results showed that the expressions of bcl-2 and MMP-9 were downregulated, and the expressions of p21 were upregulated in the MG-63 cells with overexpression of KLF6. Taken together, our results suggested that KLF6 could inhibit proliferation and invasion, and facilitate apoptosis of osteosarcoma cells, which might be a potential target for the treatment of osteosarcoma.