Forkhead box N3 (FOXN3) has been reported to be downregulated in numerous cancers, including laryngeal, oral squamous cell and hepatocellular carcinomas, and diffuse large B‑cell lymphoma. FOXN3 was proposed to serve as a tumor suppressor; however, the function of FOXN3 in osteosarcoma (OS) remains unknown. The present study suggested that FOXN3 was notably downregulated in OS tissues compared with in adjacent normal tissues, and the expression of FOXN3 was negatively correlated with tumor size, metastasis and tumor, node and metastasis stage. Additionally, low expression levels of FOXN3 predicted a poor prognosis of patients with OS. Additionally, the present study revealed that FOXN3 was also downregulated in OS cells. Numerous functional experiments, including colony formation, Cell Counting Kit‑8, wound healing and Transwell invasion assays, were performed. The results of the present study revealed that FOXN3 suppressed the proliferation, migration and invasion of OS cells. SIRT6 has been reported to serve a key role in OS; chromatin‑immunoprecipitation (ChIP) and quantitative ChIP, as well as a luciferase reporter assay, demonstrated that SIRT6 was transcriptionally regulated by FOXN3. Furthermore, FOXN3 also regulated matrix metalloproteinase‑9 secretion via the regulation of SIRT6 expression. The findings of the present study indicated that FOXN3 serves as a tumor suppressor in OS and proposed FOXN3 as a prognostic predictor and a therapeutic target for patients with OS.