Doxorubicin (Dox) is one of the most commonly used chemotherapeutic drugs for osteosarcoma (OS) treatment. In the present study, we attempted to investigate the mechanism by which Sox2OT-V7 dysregulation affects Dox chemoresistance to provide a novel experimental basis for developing neoadjuvant therapy. Sox2OT-V7 expression is upregulated in OS tissues, particularly in chemoresistant OS tissues, and in OS cell lines compared to controls. Dox treatment induces autophagy and Sox2OT-V7 expression in U2OS cells, and Dox-induced autophagy is partially attenuated by Sox2OT-V7 silencing. Knocking down Sox2OT-V7 or blocking autophagy in Dox-resistant U2OS/Dox cells resensitizes the cells to Dox treatment in vitro. Moreover, Sox2OT-V7 directly targets miR-142/miR-22 to inhibit their expression, and the effect of Sox2OT-V7 silencing on U2OS cell autophagy and U2OS/Dox cell sensitivity to Dox can be reversed by miR-142/miR-22 inhibition. Sox2OT-V7 silencing enhances the suppressive effects of Dox on U2OS/Dox cell-derived tumor growth in vivo, while miR-22 inhibition or miR-142 inhibition reverses the effects of Sox2OT-V7 silencing on Dox-induced suppression on tumor growth. Finally, miR-142 directly targets ULK1, ATG4A, and ATG5, while miR-22 directly targets ULK1 to inhibit the expression of the target gene; The Sox2OT-V7/miR-142/miR-22 axis modulates autophagy in OS cells by regulating ULK1, ATG4A, and ATG5.