The antibacterial mechanism of ε-polylysine hydrochloride (ε-PLH) against Shewanella putrefaciens was investigated at the cellular level. The inhibitory effects of ε-PLH against S. putrefaciens were determined by minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and microbial growth curve. The permeability and integrity of cell structure were determined by electrical conductivity, propidium iodide (PI)intake, alkaline phosphatase (AKPase), lactate dehydrogenase (LDHase) and adenosine triphosphatase (ATPase) activity, and scanning electron microscope (SEM) was used to evaluate the effect of ε-PLH on the cell structure of S. putrefaciens. The results showed that the MIC and MBC of ε-PLH against S. putrefaciens were 1.0 mg/mL and 2.0 mg/mL respectively. The growth of bacteria was inhibited, the extracellular AKPase and LDHase activities were increased significantly, while the ATPase activity in the cell membrane was decreased. The results of PI intake and electrical conductivity were greatly positively correlated with the concentration of ε-PLH. The results of SEM showed that after ε-PLH treatment, the bacteria presented depression, holes and other phenomena, and the appearance had changed. In conclusion, after ε-PLH treatment, the bacterial cell structure was seriously damaged, the contents leaked, and the bacterial cell shape greatly changed, and led to cell apoptosis finally. The research on the mechanism of ε-PLH against S. putrefaciens can provide the theoretical reference for the application of ε-PLH in aquatic products preservation.