OBJECTIVE To investigate the effect of polyphyllin Ⅱ (PPⅡ) on apoptosis of osteosarcoma cells and its mechanism. METHODS Osteosarcoma (OS) U2OS and HOS cell lines were selected as the research object. CCK-8 method was used to detect the effect of PPⅡ on cell viability. Colony forming assay was used to detect the effect of PPⅡ on colony formation ability. EdU incorporation assay was used to detect the effect of PPⅡ on DNA synthesis. Annexin V-FITC/PI staining assay was used to detect the effect of PPⅡ on cell apoptosis. JC-1 assay was used to determine the effect of PPⅡ on the mitochondrial membrane potential (MMP). The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cysteinyl aspartate specific proteinase 3 (Caspase 3), Caspase 7, Caspase 9, cleaved cysteinyl aspartate specific proteinase 3 (cleaved Caspase 3), cleaved Caspase 7, cleaved Caspase 9, poly (ADP-ribose) polymerase (PARP), cleaved poly (ADP-ribose) polymerase (cleaved PARP), protein kinase R like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4), p-PERK, and p-eIF2α were analyzed by Western blot. RESULTS PPⅡ significantly inhibited the viability of OS cells in a time- and dose-dependent manner, dose-dependently inhibited the colony formation and DNA synthesis, significantly promoted apoptosis rate, decreased MMP (P<0.05 or P<0.01), and increased Bax/Bcl-2 protein ratio and the protein expression levels of cleaved Caspase 3, cleaved Caspase 7, cleaved Caspase 9, cleaved PARP, p-PERK, p-eIF2α, ATF4, CHOP (P<0.05 or P<0.01). CONCLUSION PPⅡ promotes OS cell apoptosis by regulating mitochondrial pathway and mediating endoplasmic reticulum (ER) stress.