Background <p>Ultrasound is considered a safe and non-invasive tool in regenerative medicine. In particular, low-intensity pulsed ultrasound (LIPUS) has been used in the clinic for more than twenty years for applications in bone healing. It has been demonstrated to be an effective tool to treat different chronic diseases. We sought to evaluate the effects produced by LIPUS on the properties of human breast cancer stem cells (bCSCs).</p> Methods <p>Cells were stimulated using a traditional ultrasound device with the following parameters: 0.05&#xa0;W/cm² with 10% duty cycle, frequency of 3&#xa0;MHz and 8 pulses.</p> Results <p>At the parameters used, the ultrasound did not directly affect bCSC proliferation, with no evident changes in morphology. In contrast, the ultrasound protocol affected the migration and invasion ability of bCSCs, limiting their capacity to advance while a major affection was detected on their angiogenic properties. LIPUS-treated bCSCs were unable to transform into aggressive metastatic cancer cells, by decreasing their migration and invasion capacity as well as vessel formation. Finally, RNA-seq analysis revealed major changes in gene expression, with 676 differentially expressed genes after LIPUS stimulation, 578 upregulated and 98 downregulated.</p> Conclusions <p>Overall, these results highlight the potential of LIPUS as a promising non-invasive therapy to target bCSCs and attenuate its capacity to drive migration, invasion, angiogenesis and, ultimately, tumor malignancy. Besides, the ability of LIPUS to modulate gene expression points out its capacity to broadly influence the cellular transcriptome. Therefore, the application of LIPUS as an antitumor therapeutic agent targeting bCSCs may offer a promising new approach to treat cancer. In vivo functional experiments will determine in the future the relevance of LIPUS application for the development of metastasis.</p>