The aim of the present study was to evaluate the mechanism of antimicrobial action of Lysine (Lys) against Escherichia coli (E. coli) O157:H7. Lys alters the permeability and morphology of bacterial cell membranes, leading to leakage of intracellular material. Using agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), it was found that high concentrations of Lys disrupted the DNA and protein primary structure of E. coli O157:H7. Lys also inhibited the ability of E. coli biofilm formation in a concentration-dependent manner. Subinhibitory concentration of Lys reduces the swimming and swarming ability of E. coli O157:H7 and its ability to adhere and to invade Caco-2 cells. The use of Lys on lettuce inoculated with E. coli O157:H7 showed good disinfection efficiency. Further studies revealed that Lys had both preventive and therapeutic effects on the severity of colitis induced by E. coli O157:H7 infected mice, and that the preventive effect was greater than the therapeutic effect.