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Piezo1 ion channels play a crucial role in apoptosis regulation in human breast cancer cells (MCF-7), and this study evaluates the effects of Piezo1 agonist (Yoda1), inhibitor (GsMTx4), and ultrasound microbubble (USMB) treatment on cellular apoptosis pathways. In this research, in vitro cultures of normal breast epithelial cells (MCF-10A) and cancer cell lines (MCF-7, MDA-MB-231) were analyzed by Western blotting to determine Piezo1 protein levels, with MCF-7 selected for further analysis. Groups included control (untreated), Yoda1, USMB, GsMTx4, and USMB+GsMTx4, and apoptosis rates were measured via flow cytometry. Levels of apoptosis-related proteins (Bcl-2, Bax), endoplasmic reticulum stress proteins (GRP-78, Caspase 12), and mitochondrial pathway proteins (Cyt-c, Caspase 3, Caspase 9) were quantified, while JC-1 and Ca2+ fluorescent probes were used to assess mitochondrial membrane potential and intracellular Ca2+ concentration. Results showed MCF-7 cells expressed the highest Piezo1 levels. Yoda1 and USMB both markedly increased apoptosis, enhanced ER stress, and induced the mitochondrial apoptosis pathway in comparison to control, while GsMTx4 had the opposite effect and USMB reversed GsMTx4's phenotype. The USMB group exhibited the lowest mitochondrial membrane potential and the highest Ca2+ fluorescence intensity. These findings indicate that USMB activates ER stress via Piezo1, induces mitochondrial dysfunction, elevates intracellular Ca2+, and thereby promotes apoptosis in breast cancer cells.​​.