Homeobox B8 (HOXB8) is a member of HOX family and was reported to be dysregulated in human cancers. However, its expression pattern and function in human osteosarcoma (OS) remain unknown. The aim of the current study is to examine its expression and biological roles in human OS cells. Our results showed that HOXB8 was highly expressed in human OS tissues and cell lines. Knockdown of HOXB8 significantly suppressed the proliferation of OS cells in vitro and attenuated the tumor growth in a tumor xenograft model. In addition, knockdown of HOXB8 dramatically repressed the migration and invasion of OS cells. Furthermore, knockdown of HOXB8 efficiently prevented the activation of Wnt/β-catenin signaling pathway in OS cells. In conclusion, the findings of the present study demonstrated that knockdown of HOXB8 could suppress tumorigenesis and metastasis in OS through regulation of the Wnt/β-catenin signaling pathway. Thus, HOXB8 may represent a novel therapeutic target for the treatment of OS.