Abstract: D-(+) -3-Phenyllactic acid (PLA) was a novel organic acid with strong antibacterial and antifungal activities isolated from fermented food recently. In order to study the effect of PLA against L. monocytogenes 10403s on cell membrane integrity and permeability, flow cytometry, fluorescence microscopy and scanning electron microscopy were used. The results indicated that, the membrane permeation was the highest which accounted for 90.6% with flow cytometry after L. monocytogenes 10403s against with PLA (MIC) for 1 h. In addition, the integrity of cell membrane was significantly damaged with the extension of time, and the damage rate reached 91.9% after treated with PLA for 1 h. The cell membrane destruction ratios were showed by fluorescence microscopy intuitively. The microstructure of L.monocytogenes 10403s treated with PLA for 1, 3, 6 h had been determined by scanning electron microscope (SEM). The results showed that the cell wall was damaged with holes, deformation, adhesion and discoursing in group after for 6 h. PLA could affect the integrity and permeability of L.monocytogenes 10403s cell membrane, providing a theoretical basis for its good application in the field of food preservation.