Purpose <p>The expression of CD24 is significantly higher in triple-negative breast cancer (TNBC) compared to estrogen receptor and progesterone receptor-positive (ER⁺ PR⁺) breast cancer and normal breast tissue. CD24 overexpression is associated with tumorigenesis, metastasis, and drug resistance in TNBC. Moreover, CD24 functions as a “don’t eat me” signal and disrupts macrophages-mediated phagocytosis of cancer cells. We, therefore, aim to investigate the therapeutic potential of downregulating CD24 in TNBC cells.</p> Methods <p>We designed four CD24 siRNAs and evaluated their silencing efficiency and biological activity in TNBC cells using different methods. We also examined the impact of CD24 silencing on doxorubicin resistance and macrophage-mediated phagocytosis of TNBC cells.</p> Results <p>We have identified a CD24 siRNA that exhibits potent silencing activity in TNBC cells, effectively inhibiting their proliferation, migration, and invasion. CD24 silencing also induces apoptosis of TNBC cells and arrests the cell cycle in the S phase. Moreover, silencing CD24 enhances the sensitivity of TNBC cells to doxorubicin and increases macrophage-mediated phagocytosis of TNBC cells.</p> Conclusions <p>Targeting CD24 with siRNAs is a promising therapeutic strategy for TNBC and other cancers characterized by CD24 overexpression.</p>