PIEZO1 are Ca2+-permeable mechanogated channels that play a crucial role in numerous fundamental cellular responses. Ca2+ influx via PIEZO1 could control the activity of various Ca2+-dependent molecules within the cells, thus activating Ca2+-dependent signaling processes and reactions. Previously we have demonstrated Ca2+-mediated coupling between PIEZO1 and KCa channels in the plasma membrane of transformed mouse fibroblasts, where Ca2+ influx through PIEZO1 stimulates the activity of functionally co-localized KCa channels. Importantly, the selective PIEZO1 activator, Yoda1, inhibited transformed fibroblast migration, induced F-actin assembly and stress fiber formation. However, the impact of PIEZO1-KCa channel coupling to the observed effects remains unknown. Here, we performed the molecular identification of KCa channels in transformed mouse fibroblasts. Importantly, TRAM-34, a specific KCa3.1 channel blocker, abrogated the effect of Yoda1 on F-actin organization and fibroblast motility. We could conclude that KCa3.1 channels in the plasma membrane are primary downstream effectors and critical contributors to the decrease in transformed fibroblast migration and F-actin assembly caused by selective PIEZO1 activation.