Zinc finger and BTB domain-containing protein (ZBTB) proteins have been implicated in different cellular processes, including DNA damage responses and cell cycle progression. However, the mechanism by which ZBTB14 modulates radiotherapy (RT) radioresistance (RT-R) remains to be elucidated. We aimed to elucidate the regulation mechanism of ZBTB14 in breast cancer (BC) RT-R. Using integrated bioinformatics prediction, ZBTB14 was identified as a hub transcription factor related to RT-R in BC. ZBTB14 was significantly under-expressed in non-responders and RT-R/BC cells, whereas its target transmembrane protein 208 (TMEM208) was significantly overexpressed in non-responders and RT-R/BC cells. Chromatin immunoprecipitation-qPCR and luciferase reporter assays revealed that ZBTB14 downregulated TMEM208 expression through transcriptional repression. Overexpression of ZBTB14 significantly inhibited the malignant biological behavior of BC cells and tumor growth in vivo, and further upregulation of TMEM208 reversed the biological activity and radiotherapy resistance of RT-R/BC cells inhibited by overexpression of ZBTB14.