Objective To investigate whether the long non-coding RNA UNC5B-AS1 (UNC5B-AS1) regulates malignant phenotypes of osteosarcoma cells through the microRNA (miR)-373-5p/Bcl-2 interacting protein 3 like protein (BNIP3L) axis. Methods Cell proliferation, migration/invasion, and apoptosis were assessed using CCK-8 assay, transwell assay, annexin V/PI staining, and JC-1 staining. The expression of UNC5B-AS1, miR-373-5p and BNIP3L was examined by Real-time PCR, Western blotting, and immunohistochemiscal staining in osteosarcoma tissue and adjacent non-tumor tissue, as well as in hFOB1.19 cells and osteosarcoma cell lines (MG-63, 143B and U2OS). Dual-luciferase reporter assay was performed to validate the direct targeting of BNIP3L by miR-373-5p. A nude mouse xenograft model was used for in vivo validation. Results UNC5B-AS1 was highly expressed in MG-63 and 143B cells, and its knockdown significantly increased miR-373-5p levels. Overexpression of miR-373-5p inhibited proliferation and migration/invasion and promoted apoptosis, accompanied by a decrease in mitochondrial membrane potential. BNIP3L and the microtubule-associated protein 1A/1B light chain 3（LC3Ⅱ/LC3Ⅰ）ratio were reduced while ubiquitin-binding protein p62 (P62) increased, suggesting suppressed mitophagy. Dual-luciferase assay confirmed that miR-373-5p directly targets and inhibits BNIP3L. BNIP3L overexpression partially rescued the inhibitory effects of miR-373-5p on proliferation and invasion. In vivo, BNIP3L overexpression promoted tumor growth, whereas miR-373-5p partially attenuated this effect. Conclusion UNC5B-AS1 could modulates osteosarcoma cell proliferation, migration/invasion, and apoptosis via the miR-373-5p/BNIP3L signaling axis.