Sodium metasilicate (SMS) is a U.S. Department of Agriculture-approved antimicrobial for use in meat and poultry processing and has been known to be effective against various foodborne pathogens. However, its antimicrobial mechanism has not yet been revealed. In this study, we attempted to elucidate the mechanism by which SMS inactivates Listeria monocytogenes, a Gram-positive bacterial pathogen encountered commonly in ready-to-eat meat and poultry products. L. monocytogenes (Scott A) cells were treated with different concentrations of SMS (1.0, 2.0, 3.0, 4.0, 5.0, and 6.0% [wt/vol]) and compared with high pH treatment (0.1, 0.2, and 0.3N NaOH solutions) for 1, 10, and 30 min. SMS exhibited concentration and time effects on inactivation of L. monocytogenes. The effect of SMS on the membrane integrity and viability of L. monocytogenes was determined by use of propidium iodide (PI) and SYTO9 nucleic acid stains with subsequent flow cytometry. The breakage in membrane integrity was observed by uptake of PI by cells treated with SMS with subsequent flow cytometry. Ultrastructural changes from corresponding transmission electron micrographs further revealed the disruption in the cytoplasmic membrane and changes in the morphology of the cells treated with SMS and high pH. The results from flow cytometry experiment and transmission electron microscopy study indicated that following SMS treatment, the membrane integrity of L. monocytogenes was compromised leading to leakage of intracellular contents and subsequent cell death.