The aim of this work was to investigate the mechanisms underlying the inactivation of Listeria monocytogenes in raw milk under high hydrostatic pressure (HHP). The results showed that a 450-MPa treatment for 5 min could completely eliminate 10⁸ colony-forming units/mL of viable bacteria, as indicted by increased uptake of propidium iodide. Morphological damage to the cell wall, cell membrane, and cytoplasmic components by HHP treatments was observed on scanning and transmission electron micrographs. The protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels differed between HHP-treated and untreated L. monocytogenes, in that HHP decreased the protein content and caused partial protein degradation. Therefore, our results indicate that HHP inactivates L. monocytogenes by causing morphological changes in the internal and external cellular structures, as well as through membrane damage, cell wall rupture, and membrane protein degradation.