Background: Osteosarcoma (OS), the most prevalent primary bone cancer, disproportionately impacts children and adolescents, significantly affecting their physical capabilities and psychological well-being. MicroRNAs (miRNAs), including miR-377, have been recognized for their pivotal roles in the oncogenesis and progression of various cancers. This study delves into the influence of miR-377 on OS development, with a particular emphasis on its implications for athletic performance and mental health in affected individuals. Methods: Quantitative RT-PCR (qRT-PCR) was utilized to measure the expression levels of miR-377 and its target gene CUL1 in OS tissues and cell lines. The effects of miR-377 on OS cell proliferation, invasion, and migration were assessed using MTT and transwell assays. Luciferase reporter assays confirmed CUL1 as a direct target of miR-377. Western blot analysis was conducted to explore the impact of miR-377 on the epithelial-mesenchymal transition (EMT) and the Wnt/β-catenin signaling pathway. Additionally, tumor xenograft models were employed to investigate the in vivo effects of miR-377 on OS tumorigenesis. Results: miR-377 was found to be significantly downregulated in OS tissues and cell lines, correlating with a poorer prognosis for OS patients. Restoration of miR-377 expression markedly inhibited OS cell growth, invasion, and migration by modulating the EMT process and the Wnt/β-catenin signaling pathway. Luciferase reporter assays further confirmed CUL1 as a functional target of miR-377, with elevated CUL1 expression in OS tissues also indicative of poor patient outcomes. In vivo studies demonstrated a substantial reduction in OS tumor growth following miR-377 overexpression. Conclusion: The findings from this study underscore the critical role of miR-377 in suppressing OS progression through targeting CUL1 and modulating the EMT and Wnt/β-catenin signaling pathways. By elucidating these mechanisms, our research highlights the potential of miR-377 as a therapeutic target in OS treatment, offering hope for improved recovery outcomes, enhanced athletic performance, and better mental health for individuals afflicted by this disease. These insights pave the way for the development of miR-377-based interventions that could significantly benefit patients' quality of life by addressing both the physical and psychological challenges posed by OS.