Purpose <p>Reduced response to targeted therapy in human epidermal growth factor receptor 2 (HER2)-positive breast cancer underscores the need for novel therapeutic strategies and molecular targets. Cluster of differentiation 24 (CD24), an oncogenic molecule closely associated with aggressive tumor phenotypes, was investigated for its expression in HER2-positive breast cancer and its potential as a candidate target for combination therapy.</p> Methods <p>Herein, we explored the expression of CD24 in breast cancer (BC) and its prognostic significance. In HER2-positive BC cells, CD24 was knocked down to assess its impacts on tumor biological behaviors and synergistic effects with trastuzumab. Furthermore, the underlying mechanism of CD24 was elucidated using a series of experimental assays, including Western blotting (WB), immunohistochemistry (IHC), terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and mRFP-GFP-LC3 dual-fluorescence autophagy assay. In vivo experiments were conducted to evaluate the therapeutic efficacy of CD24 inhibition combined with trastuzumab.</p> Results <p>CD24 was highly expressed in HER2 + breast cancer and associated with poor prognosis. CD24 knockdown significantly suppressed the proliferation, migration, and invasion of HER2-positive BC cells. Moreover, CD24 depletion promoted apoptosis and autophagy in HER2-positive breast cancer, accompanied by the inactivation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway. In vivo data demonstrated that the combination regimen of CD24 knockdown and trastuzumab markedly reduced tumor burden.</p> Conclusion <p>CD24 silencing modulates autophagy and induces apoptosis via Akt/mTOR inhibition, and its combination with trastuzumab exerts potent antitumor effects, offering a promising strategy for HER2 + breast cancer.</p>