Osteosarcoma is a malignancy that primarily affects children and young adults. The poor survival is largely attributed to acquisition of chemoresistance. Thus, the current study aimed to elucidate the role of ELK1/miR-134/PTBP1 signaling cascade in osteosarcoma chemoresistance. Doxorubicin (DXR)-resistant human osteosarcoma cells were initially self-established by continuous exposure of MG-63, U2OS and HOS cells to increasing DXR doses. Osteosarcoma chemoresistance in vitro was evaluated using CCK-8 assays and EdU staining. Aerobic glycolysis was evaluated by lactic acid production, glucose consumption, ATP levels, and Western blot analysis of GLUT3, HK2 and PDK1 proteins. The nude mice were injected with 5.0 mg/kg DXR following the subcutaneous transplantation of osteosarcomas. PTBP1 was upregulated in tumor tissues derived from non-responders to DXR treatment and correlated with patient poor survival. PTBP1 enhanced chemoresistance in cultured osteosarcoma cells in vitro and in vivo by increasing aerobic glycolysis. Additionally, miR-134 inhibited translation of PTBP1. ELK1 bound to miR-134 promoter and inhibited its expression. Overexpressed ELK1 enhanced chemoresistance and increased aerobic glycolysis by downregulating miR-134 and upregulating PTBP1 in DXR-resistant cells. Altogether, the key findings of the present study highlight ELK1/miR-134/PTBP1 signaling cascade as a novel molecular mechanism underlying the acquisition of osteosarcoma chemoresistance.