Despite more effective chemotherapy combined with limb-salvage surgery for the osteosarcoma treatment,survival rates for osteosarcoma patients have stagnated over the past three decades due to the poor prognosis.Osteosarcoma cancer stem cells (OSCs) are responsible for the growth and metastasis of osteosarcoma.The existence of OSCs offers a theoretical explanation for therapeutic failures including tumor recurrence,metastasis,and drug resistance.Understanding the pathways that regulate properties of OSCs may shed light on mechanisms that lead to osteosarcoma and suggest better modes of treatment.In this study,we showed that the expression level of Kruppel-like factor 4 (KLF4) is highly associated with human osteosarcoma cancer stemness.KLF4-overexpressed osteosarcoma cells displayed characteristics of OSCs:increased sphere-forming potential,enhanced levels of stemness-associated genes,great chemoresistance to adriamycin and CDDP,as well as more metastasis potential.Inversely,KLF4 knockdown could reduce colony formation in vitro and inhibit tumorigenesis in vivo,supporting an oncogenic role for KLF4 in osteosarcoma pathogenesis.Furthermore,KLF4 was shown to activate the p38 MAPK signaling pathway to promote cancer stemness.Altogether,our studies uncover an essential role for KLF4 in regulation of OSCs and identify KLF4-p38 MAPK axis as a potential therapeutic target for osteosarcoma treatment.