Background <p>Osteosarcoma (OS) is one of the more common malignant tumors, with a poor prognosis for patients that seriously affects their quality of life.</p> Objectives <p>This study examined the relationship between MCM3AP-AS1 and the severity and prognosis of OS patients, and investigated its impact on OS progression by targeting miR-205-5p.</p> Methods <p>Quantitative PCR to detect the expression of MCM3AP-AS1 and miR-205-5p in OS tissues and cells. The clinical relevance of MCM3AP-AS1 was analyzed using a Chi-square test. Risk factors related to poor prognosis and recurrence-free survival rate were analyzed using the Cox risk model and the K–M curve. The dual luciferase assay and/or RIP assay were performed to confirm the targeting relationship between MCM3AP-AS1 and miR-205-5p, as well as YAP1 and miR-205-5p. The impact of MCM3AP-AS1 targeting miR-205-5p/YAP1 axis on OS progression was analyzed using CCK-8 and Transwell functional experiments.</p> Results <p>MCM3AP-AS1 was significantly increased in OS tissues/cells, correlating with adverse clinicopathological features and poor prognosis. miR-205-5p was down-regulated and negatively correlated with MCM3AP-AS1. Dual luciferase reporter and/or RIP assays confirmed that MCM3AP-AS1 directly targeted miR-205-5p, and miR-205-5p directly bound to the 3’UTR of YAP1. Silencing MCM3AP-AS1 inhibited OS cell functions, while reducing mRNA and protein expression of YAP1. Co-inhibiting miR-205-5p reversed these effects and restored YAP1 expression. Triple intervention reversed the promotion of cell malignant behaviors induced by dual intervention.</p> Conclusion <p>MCM3AP-AS1 promotes OS via sponging miR-205-5p/YAP1 axis, serving as a potential prognostic marker and candidate therapeutic target.</p>