Invasiveness research is an essential step in breast cancer metastasis. The application of high-frequency ultrasound microbeam stimulation (HFUMS) offers a manner of determining the invasion potential of human breast cancer cells by eliciting the elevation of transient cytoplasmic calcium ions (Ca2+). The fluorescent change in invasive breast cancer cells (MDA-MB-231), reflecting Ca2+ elevation elicited by HFUMS, is greater compared to that in weakly invasive breast cancer cells (MCF-7). GsMTx4, a selective blocker of mechanosensitive Piezo channels, was used for better understanding of mechanotransduction in HFUMS on cells.