It is known that only listeriolysin O (LLO)-producing Listeria monocytogenes strains are able to induce protective immunity, but the underlining relationship between LLO produced by virulent strains and generation of protective immunity in the infected host remains poorly understood. In the present study, it was found that LLO gene expression was only detected in the mice infected with virulent strain which was able to induce protective immunity, while non-virulent strains or killed bacteria were not able to generate protective immunity. When mice were immunized with LLO plus killed bacteria in the presence of incomplete Freund's adjuvant, the protective immunity was partially generated, and adoptive transfer experiment confirmed that this protection was antigen specific. Reverse-transcription polymerase chain reaction revealed that LLO plus killed bacteria induced the expression of interferon-gamma (IFN-gamma) and interleukin-12 (IL-12). Our results also showed CD4+ T cells were the principal cells constituting protective immunity. Taken together, it may be concluded that LLO produced from virulent strains of L. monocytogenes was essential for the generation of protective immunity, and that LLO plus killed bacteria induced IFN-gamma and IL-12 expression which resulted in the generation of protective immunity.