Increased expression of cancer/testis antigens (CTAs) is reported in various tumors. However, the unique role of CTAs in tumor genesis has not yet been verified. Here, we first report the functional role of CT45A1 in the carcinogenesis of osteosarcoma. RNA sequencing and immunohistochemistry confirmed that elevated expression of CT45A1 was detected in osteosarcoma, especially in metastatic tissues of osteosarcoma. Furthermore, osteosarcoma patients with poorer prognosis showed high expression of CT45A1. In cell tests, CT45A1 overexpression was shown to strengthen the proliferation, migration, and invasion abilities of osteosarcoma cells, while silencing CT45A1 markedly elicited the opposite effects in these tests by disrupting the activation of β-catenin. In summary, we identify a novel role of CT45A1 in osteosarcoma. Furthermore, our results suggested that CT45A1 may contribute to the development of osteosarcoma and could be a possible therapeutic target for osteosarcoma patients.