Adenoid cystic carcinoma (AdCC) is highly metastatic and resistant to chemotherapy and radiotherapy. Recently, we reported that the T-box transcription factor Brachyury is a potential regulator of cancer stem cells (CSCs). Specifically, growth of CSCs was found to be controlled by Brachyury knockdown in AdCC. Since CSCs are resistant to chemotherapy and radiotherapy, this finding provides a new principle for therapies targeting CSCs. In the present study, we established that Brachyury knockdown suppresses chemoresistance and radioresistance in vitro. Brachyury was knocked down by transfecting Brachyury short hairpin RNA (shRNA) into the AdCC CSC cell line ACCS-M GFP. Brachyury knockdown significantly inhibited cell migration and invasion and suppressed chemoresistance. A quantitative PCR array of drug transporter genes revealed that knockdown of Brachyury caused down-regulation of ATP-binding cassette transporter genes. Furthermore, ACCS-M GFP radioresistance was significantly suppressed by Brachyury knockdown. Knockdown of Brachyury significantly sensitized ACCS-M GFP cells to chemoradiotherapy. This study demonstrates that Brachyury knockdown reduces invasiveness and chemoresistance and radioresistance of CSCs in vivo. Therefore, Brachyury knockdown may be a useful therapeutic tool for sensitizing CSCs to conventional chemoradiotherapy.