The transcript factor LHX2 is dysregulated in many cancers but its role in osteosarcoma (OS) remains unclear. In this study, we confirm that LHX2 is up-regulated in osteosarcoma, and that its silencing inhibits OS malignancy and induces autophagy via mTOR signaling. We further demonstrate that miR-129-5p negatively regulates LHX2 and suppresses the malignant phenotypes of OS. LHX2 overexpression could restore the malignant phenotypes. In conclusion, LHX2 regulates tumorigenesis and autophagy via mTOR in OS and is negatively regulated by miR-129-5p. Targeting the miR-129-5p/LHX2/mTOR axis therefore represents a novel therapeutic strategy for OS treatment.