Hydroxygenkwanin (HGK) has an anticancer effect in a variety of tumors, but its role in osteosarcoma has not been explored. The purpose of the present study was to investigate the therapeutic effect of HGK on osteosarcoma and its specific molecular mechanism. Osteosarcoma cells (MG-63 and u2oS) treated with various concentrations of HGK were assigned to the treatment group. MTT, clone formation, wound healing and Transwell assays were performed to assess the viability, proliferation, migration, and invasion of MG-63 and u2oS cells. rT-qPcr was conducted to quantify the expression levels of of microRNA (miR)-320a and SRY-box transcription factor 9 (SOX9) in MG-63 and U2OS cells. The binding sites of miR-320a and SOX9 were predicted by star-Base database, and verified using the dual-luciferase reporter assay. The expression levels of SoX9 and eMT-related proteins (n-cadherin, e-cadherin and vimentin) were detected by western blot analysis. HGK inhibited cell proliferation, migration, invasion, but promoted the expression of miR-320a in MG-63 and U2OS cells. Downregulation of miR-320a reversed the effects of HGK on proliferation, migration and invasion of MG-63 and U2OS cells, while upregulation of miR-320a had the opposite effect. HGK inhibited the expression of SoX9 by promoting the expression of miR-320a. Upregulation of SOX9 could partially reverse miR-320a-induced migration and invasion of MG-63 and U2OS cells. In addition, upregulation of miR-320a promoted E-cadherin expression and inhibited the expression of n-cadherin and vimentin, and the effect of miR-320a was also reversed by SOX9. In conclusion, HGK inhibited proliferation, migration and invasion of MG-63 and U2OS cells through the miR-320a/SOX9 axis.