<p>Osteosarcoma (OS) is a common malignancy of the bone that originates from stromal cell lines. One of the key cellular pathways extensively studied in OS is the mitogen-activated protein kinase (MAPK) pathway, particularly ERK1/2, whose activation is closely associated with tumor growth and metastasis. MicroRNA (miRNA)-based detection and targeted therapies offer promising new strategies for the treatment of OS. In this study, we investigated the role of miR‑486‑3p in the regulation of the ERK1/2 pathway in OS. We examined the expression level of miR‑486‑3p in the GEO dataset (GSE65071) and clinical samples, and analyzed its regulation of the target gene SPRED1 in OS cells and tumor-bearing mice. Downregulation of miR‑486‑3p was confirmed in OS tissues, with its expression decreasing in line with the progression of clinical stages. Furthermore, the exogenous introduction of a miR-486-3p mimic attenuated the malignant behavior of OS cells, inhibiting their proliferation, migration, and invasion. Bioinformatic analysis revealed that miR‑486‑3p directly targets SPRED1 in OS, leading to alterations in epithelial-to-mesenchymal transition (EMT) markers, including E-cadherin, N-cadherin, and Vimentin. Functional loss- and gain-of-function experiments confirmed that miR‑486‑3p directly targets SPRED1 and inactivates the ERK1/2 pathway in both OS cells and tumor-bearing mice. This review demonstrates that downregulation of miR-486-3p leads to increased SPRED1 expression, which activates the ERK1/2 pathway in OS. Targeting miR-486-3p and SPRED1 could offer potential therapeutic benefits.</p> Graphical abstract <p></p>