In order to obtain an attenuated vaccine candidate for enteropathogenic Escherichia coli (EPEC) O45, a ler deletion mutant of pig enteropathogenic E. coli (PEPEC) O45 was constructed by using the suicide vector pCVD442, termed as PEPEC O45(Dler). The culture supernatant of PEPEC O45(Dler) deletion mutant was inoculated in vero cell culture. PEPEC O45(Dler) deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the PEPEC O45(Dler) deletion mutant and the virulent strain O45 respectively. Mice were observed daily for clinical signs and weight changes. Test group of mice inoculated with PEPEC O45(Dler) gained weight normally and experienced no clinical signs. In contrast, control group of mice inoculated with virulent strain O45 exhibited weight loss and all died in four days. In another experiment, pregnant mice and pig were orally vaccinated by PEPEC O45(Dler) twice at interval of 14 days respectively. Subsequently, the suckling mice and pig were orally challenged with O45 at 7 days of age respectively. The results showed that 80% of the sucking mice born by vaccinated mice and 75% of the sucking pig born by vaccinated pig were survival; 15% of the sucking mice born by non-vaccinated mice and 10% of the sucking pig born by non-vaccinated pig were survival. This study demonstrated that PEPEC O45(Dler) deletion mutant lost the toxigenicity to vero cell and to be safety to mice and pig. Oral immunization can induce specific immune responses in mice and pig, and this mutant strain could be used as an attenuated vaccine candidate against PEPEC O45.