Transmembrane protein 45B (TMEM45B) is a member of the TMEM family of proteins and has been reported to be expressed abnormally in different kinds of human tumors. However, the biological function of TMEM45B in osteosarcoma remains unclear. The objective of this study was to investigate the role of TMEM45B in regulating the biological behavior of osteosarcoma cells. Our results demonstrated that the expression of TMEM45B at both the protein and mRNA levels was dramatically upregulated in human osteosarcoma cell lines. Knockdown of TMEM45B significantly suppressed the proliferation, migration, and invasion of U2OS cells in vitro. Mechanistically, knockdown of TMEM45B sharply downregulated the expression level of β-catenin, cyclin D1, and c-Myc in U2OS cells. Finally, knockdown of TMEM45B attenuated tumor growth in transplanted U2OS-derived tumors in nude mice. Taken together, our results demonstrated that TMEM45B plays an important role in regulating the proliferation, migration, and invasion of osteosarcoma cells and that its effects on proliferation and invasion were mediated partially through the Wnt/β-catenin signaling pathway. These observations support our belief that TMEM45B may serve as an oncogene in the development and progression of osteosarcoma.