Despite improvements in the prognosis of osteosarcoma patients, chemotherapy fails in a considerable number of cases due to drug resistance. The development of novel agents may enhance chemosensitivity. This study explored the anticancer function of polydatin and its ability-in combination with paclitaxel-to overcome drug resistance in human osteosarcoma U-2OS and MG-63 cell lines. A cell proliferation assay (celll counting kit-8), a cell-cycle assay, an apoptosis assay (annexin V-fluorescein isothiocyanate/propidium iodide), and a cell migration assay (Transwell) were used to analyze cell activity. Western blot analysis and quantitative reverse-transcription polymerase chain reaction were performed to examine function-related mRNA and protein levels. Treatment with polydatin suppressed cell growth and migration in both cell lines. Moreover, polydatin induced cell apoptosis in both parental and paclitaxel-resistant cells, and cell-cycle arrest in the S phase. Furthermore, it altered the expression of various proteins associated with cell growth (Ki67, p21, cyclin A, cyclin E, and cyclin-dependent kinase 2), migration (matrix metalloproteinase-2 [MMP-2], MMP-9, and tissue inhibitor of metalloproteinase-1), apoptosis (poly[ADP-ribose] polymerase 1 and caspase 3), and drug resistance (p-glycoprotein 1, lung resistance-related protein 1, growth arrest and DNA damage-45 alpha, glutathione S-transferase pi, and heat shock protein 27) in paclitaxel-resistant osteosarcoma cells. Cells transfected with myr-Akt caused obvious upregulation and activation of Akt, which were significantly attenuated via polydatin treatment. In conclusion, polydatin may enhance the chemosensitivity of osteosarcoma cells to paclitaxel.