Most anticancer therapies trigger apoptosis to eliminate malignant cells. However, the majority of malignant cancer cells are resistant to apoptosis due to genetic mutations or heterogeneity. Here, we report that opening the pore of the bacterial large conductance mechanosensitivity channel (MscL) provides a novel approach of inducing non-apoptotic cell death. The gain-of-function mutant V23A-MscL and chemically responsive mutant G26C-MscL can be functionally expressed in hepatocellular carcinoma HepG2 cells. V23A-MscL spontaneously opens, and G26C-MscL also responds to its chemical activator MTSET. Opening of the MscL channel causes increased intracellular Ca2+ concentration and suppressed cell growth and viability. MTSET-activated G26C channels induce necrosis, while V23A-MscL expression leads to cytoplasmic vacuolization cell death in HepG2 cells and suppresses tumor growth in a mouse model. We propose that MscL may act as a nanovalve through which intracellular homeostasis suffers a disruption and results in malignant tumor cell damage, leading to a new strategy for cancer therapy. Copyright © 2020 Elsevier Ltd. All rights reserved.