As the most malignant breast cancer subtype, triple-negative breast cancer (TNBC) does not haveeffective targeted therapies clinically to date. As a selective Sp1 inhibitor, Mithramycin A (MIT) has beenreported to have anti-tumor activities in multiple cancers. However, the efficacy and the mechanismof MIT in breast cancer, especially TNBC, have not been studied. In this study, we demonstrated thatMIT suppressed breast cancer cell survival in a dosage-dependent manner. Interestingly, TNBC cellswere more sensitive to MIT than non-TNBC cells. MIT inhibited TNBC cell proliferation and promotedapoptosis in vitro in time- and dosage-dependent manners. MIT suppressed TNBC cell survival, at leastpartially, by transcriptionally down-regulating KLF5, an oncogenic transcription factor specificallyexpressed in basal TNBC. Finally, MIT suppressed TNBC cell growth in a xenograft mouse model. Takentogether, our findings suggested that MIT inhibits basal TNBC via the Sp1/KLF5 axis and that MIT maybe used for TNBC treatment.