Objectives <p>The pathogenesis of osteosarcoma remains inadequately understood. This study aims to investigate the role and underlying mechanisms of miR-301a-3p in osteosarcoma progression, providing a scientific basis for the development of effective therapeutic strategies.</p> Methods <p>Osteosarcoma tissues and cells were collected and miR-301a-3p expression was detected by qRT-PCR and assessed the correlation between miR-301a-3p expression and clinical disease and prognosis of osteosarcoma patients. The effects of miR-301a-3p overexpression on osteosarcoma cells were investigated by Transwell assay and CCK-8 assay. The targeting relationship between miR-301a-3p and MAFB was detected by dual luciferase reporter assay.</p> Results <p>miR-301a-3p was significantly downregulated in osteosarcoma. Significant differences in TNM staging and lung metastasis were observed between the high and low miR-301a-3p expression groups, with a higher mortality rate in the low expression group, indicating it as a risk factor for poor prognosis. Upregulation of miR-301a-3p inhibited the proliferation and metastasis of osteosarcoma cells. MAFB was identified as a target gene of miR-301a-3p, capable of reversing the inhibitory effects of miR-301a-3p on cancer cells.</p> Conclusions <p>Low expression of miR-301a-3p may serve as a warning sign for disease progression and poor prognosis in osteosarcoma patients. miR-301a-3p may inhibit malignant behaviors of osteosarcoma cells by targeting MAFB.&#xa0;Future studies could explore the integration of ultrasound-targeted microbubble destruction technology to enhance the targeted delivery of miR-301a-3p mimics or MAFB inhibitors, potentially overcoming limitations in drug penetration and bioavailability observed in conventional therapies.</p>