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.