Background <p>Triple-negative breast cancer (TNBC) is characterized by its aggressive nature and limited treatment options, which contribute to a poor prognosis. However, the potential role of microRNA-563 (miR-563) in this malignancy remains unclear. This study investigates the tumor-suppressive role of miR-563 and its underlying mechanisms, proposing its potential as a novel biomarker in TNBC.</p> Methods <p>The expression of miR-563 was analyzed in clinical samples of TNBC in comparison to healthy controls and non-TNBC cases. Functional assays were conducted using TNBC cell lines. Bioinformatics prediction and luciferase reporter assays identified integrin subunit alpha-V (ITGAV) as a direct target, which was further validated through rescue experiments. Additionally, epithelial-mesenchymal transition (EMT) markers were assessed to elucidate the mechanistic pathway.</p> Results <p>MiR-563 was significantly downregulated in TNBC patients, demonstrating an area under the curve (AUC) of 0.895 for diagnosis. This downregulation correlated with advanced disease stage, lymph node metastasis, and poorer progression-free survival. Overexpression of miR-563 resulted in the suppression of TNBC cell proliferation, migration, and invasion. ITGAV was identified as a direct target of miR-563, and rescue experiments indicated a partial reversal of the tumor-suppressive effects of miR-563. Furthermore, miR-563 inhibited EMT, as evidenced by the upregulation of E-cadherin and downregulation of Vimentin.</p> Conclusions <p>These findings identify miR-563 as a crucial tumor suppressor and a potential diagnostic predictor in TNBC, operating in part through the ITGAV-mediated pathway.</p>