The peroxisome proliferator-activated receptor γ (PPARγ) is emerging as an important regulator in various metabolic processes of cancer. Genistein, as a major isoflavonoid isolated from dietary soybean, possesses a wide variety of biological activities, particularly, in cancer prevention. However, the mechanisms by which genistein elicits its growth inhibiting effects in osteosarcoma (OS) MG-63 cells have not been extensively elucidated. MG-63 cells were treated for 2 days with various concentrations of genistein and/or GW9662 (a selective antagonist of PPARγ). The effect of different drugs on cell viability was determined by Cell Counting Kit-8 (CCK-8). The assay of cell proliferation was performed using 5-ethynyl-2'-deoxyuridine (EdU). The changes of apoptosis and cell cycle progression were detected by flow cytometry experiments. The protein expression of PPARγ pathway (PPARγ, PTEN, BCL-2, Survivin, P21WAF1/CIP1 and Cyclin B1) was determined by western blot analysis. The expression of PPARγ and PTEN mRNA was detected by real-time quantitative RT-PCR analysis. We report that genistein caused OS cell growth inhibition. We found that the PPARγ expression in OS cells increased after genistein treatment. Further studies on the mechanisms of genistein revealed a series of cell growth changes related to the PPARγ pathway; while cell cycle changes can be reversed by GW9662. Genistein plays an important role in preventing OS cell growth, which can impede the OS cell cycle as a non-toxic activator of PPARγ, providing novel insights into the mechanisms of the therapeutic activities of genistein.