Among all breast cancer subtypes, triple-negative breast cancer (TNBC) has the worst prognosis due to its resistance to treatment, including radiotherapy. The purpose of this study was to screen and validate potential targets related to radioresistance in TNBC. First, a radiation-resistant TNBC cell model, was generated using BT549 cells through fractionated radiation (30 Gy in total, 10 fractions). Then, mRNA sequencing was used to screen potential target genes related to radioresistance. Furthermore, bioinformatics, proteomics and genomics methods were used to screen and validate the key target genes. The results showed that BT549 cells that had undergone fractionated radiation, namely, BT549R cells, had greater proliferation and radioresistance and a lower apoptosis rate than parental BT549 cells. C-type lectin domain family 3 member B (CLEC3B) was identified as a potential key target gene related to radioresistance. The expression of CLEC3B was closely related to the radioresistance of breast cancer and the survival of breast cancer patients. Downregulation of CLEC3B increased cell viability and radioresistance and reduced apoptosis in BT549R cells, which could be reversed by dihydroartemisinin (DHA). The overexpression of CLEC3B with or without DHA treatment inhibited the activity of the PI3K/AKT signaling pathway. DHA increased the expression of CLEC3B in a concentration-dependent manner. In summary, our study illustrated that the downregulation of CLEC3B increased radioresistance in BT549 breast cancer cells and that this effect could be reversed by DHA.