<p>Transformed and cancer cells display reduced cellular stiffness, disorganized cytoskeleton and dysregulated cell–matrix adhesion, which together facilitate cell migration and invasion. Oncogenic Ras is well known for driving uncontrolled cytoskeleton remodeling. However, although cancer cells are more vulnerable to dynamic mechanical stress than normal epithelial cells and ultimately undergo apoptosis, it remains unclear whether and how Ras-transformed cells are responsive to cyclic stretch-mediated cell death. Here, we applied uniaxial cyclic stretch to an inducible H-Ras^V12-overexpressing Madin-Darby Canine Kidney (MK4) epithelial cell line and investigated its effects on Ras-transformed cell viability. Cyclic stretch induced strain-dependent detachment of H-Ras^V12-overexpressing cells prior to apoptosis, accompanied by downregulation of PCNA, c-Jun and phosphorylated focal adhesion kinase, and upregulation of the mechanosensitive calcium channel Piezo1. Short-term cyclic stretch markedly increased cytosolic calcium influx, whereas silencing Piezo1 restored cell adhesion and viability in H-Ras&#xa0;^V12-overexpressing cells, indicating that Piezo1 mediates downstream signaling events leading to cyclic stretch-induced cell detachment and apoptosis. Furthermore, reducing cytosolic calcium levels with BAPTA-AM or inhibiting calpain activity also rescued adherent H-Ras^V12-overexpressing cells from cell floating under cyclic stretch. Taken together, H-Ras^V12-transformed epithelial cells showed impaired actin-focal adhesion machinery, and cyclic stretch selectively induces cell detachment prior to apoptosis in H-Ras-overexpressing cells through a Piezo1-calpain-mediated mechanotransduction pathway.</p>