Background <p>The stemness of tumor cells is closely associated with immune infiltration and may influence both the prognosis of osteosarcoma and alterations in the tumor microenvironment.</p> Methods <p>Through single-cell RNA sequencing (scRNA-seq), we identified tumor cells exhibiting high stemness and developed a prognostic model leveraging their differentially expressed genes (DEGs). The differences between the high-risk and low-risk score groups were analyzed in terms of mutation landscapes, enriched pathways, immune cell infiltration, and immunotherapy response. Subsequently, a cross-enrichment analysis was conducted on prognostic genes, genes highly expressed in high-risk group, and DEGs of the high-stemness cluster to identify commonly upregulated gene. Functional validation of the gene was carried out in osteosarcoma cell lines MG-63 and HOS.</p> Results <p>A cluster of cancer cells with high-stemness characteristics was identified in osteosarcoma. The stemness risk score, based on genes highly expressed in the high-stemness cluster, was found to be an independent prognostic factor for osteosarcoma, capable of effectively predicting patient outcomes, and was closely related to immune infiltration. By taking the intersection of prognostic genes, genes highly expressed in the high-risk group, and DEGs from the high-stemness cluster, the gene S100A13 was identified as a key gene of stemness. Functional validation through knockdown experiments demonstrated that S100A13 is a critical factor for osteosarcoma cell proliferation and stemness, as confirmed by colony formation and tumor sphere formation assays. Interestingly, wound healing, Transwell migration and invasion assays also showed that S100A13 is associated with the malignancy of osteosarcoma cells.</p> Conclusions <p>Our study demonstrates the presence of a cluster of cells with high-stemness characteristics in the tumor microenvironment of osteosarcoma. An effective prognostic model was established based on DEGs of the high-stemness cell cluster, and S100A13 was identified as a core gene through cross-enrichment analysis. Experimental validation confirmed that S100A13 plays a critical role in the malignant progression and stemness regulation of osteosarcoma. Therefore, S100A13 is not only a key gene in the stemness risk score but also a promising biomarker for prognosis and a potential therapeutic target for osteosarcoma, and it may facilitate the development of novel diagnostic and the therapeutic strategies to improve patient outcomes in osteosarcoma.</p>