Propionic acid (PPA), utilized in the manufacture of cellulose acetate propionate, is known to exhibit antimicrobial effects, but its mechanism in Escherichia coli O157 is still unknown. In general, antimicrobial activity is associated with reactive oxygen species (ROS), but ROS generation is not observed under PPA treatment. In addition to ROS, experiments were conducted to observe changes in trehalose and ion balance to discover factors that may affect the cell proliferation. Bacteria use trehalose, a sugar used for stabilization due to stress factors, which contradicts PPA concentration. Discrepancy in homeostasis follows as a result of ion imbalance. PPA causes interruption in bacterial internal stability in a dose-dependent manner. Membrane damage by ion imbalance occurs due to the binding ability of ionized PPA and divalent ions, which induce membrane depolarization, leading to a reduction in cell viability. Considering the lethal impact of membrane depolarization on cell death in bacteria, DNA fragmentation and phosphatidylserine exposure in apoptosis are confirmed. Due to severe damage in DNA, the activation of caspase-like protein is observed. Apoptosis-like death (ALD), a novel programmed cell death in bacteria, occurs eventually. In conclusion, ALD in E. coli O157 is induced via the contribution of homeostasis disruption in a ROS-independent manner.