Breast cancer (BC) is the most prevalent malignancy among women worldwide. Despite the widespread use of chemotherapy, resistance remains a major challenge, leading to treatment failure and poor prognosis. The DNA damage response (DDR) plays a crucial role in determining cancer outcomes following chemotherapy, making it a promising therapeutic target. Interferon γ-inducible protein 16 (IFI16) functions as an innate immune sensor of double-stranded DNA. Previously, we showed that IFI16 inhibits DNA double-strand break (DSB) repair, leading to type I IFN-mediated antitumor immune response. However, its tumor-intrinsic role in DNA repair and chemosensitivity remains unclear. Here, we showed that IFI16 increased cell-intrinsic apoptosis and chemosensitivity in BC cells. Mechanistically, IFI16 interacted with the MRN complex upon DNA damage, disrupting its interaction with ATM, a key step in DDR process. In vivo xenograft experiments using BC control and IFI16-knockout (KO) cells demonstrated that doxorubicin effectively suppressed tumor growth in control cells, whereas IFI16 KO tumors exhibited reduced sensitivity to the treatment. Furthermore, analysis of publicly available datasets revealed that BC patients with high IFI16 expression had significantly higher pathological complete response rates than those with low IFI16 expression. Together, our findings suggest that IFI16 disrupts MRN complex-ATM interactions, impairing DSB repair and enhancing chemosensitivity.