The pathogenesis associated with the 36 Megadalton (Md) plasmid of Salmonella choleraesuis subsp. choleraesuis serovar Enteritidis (S. Enteritidis) was assessed by using the plasmid-containing strain AL1190, plasmid-cured strain AL1192, and plasmid-reintroduced strain AL1193. After oral inoculation of strain AL1190 or AL1192 to C57BL/6 mice, mesenteric lymph nodes, spleens, and livers were examined for the numbers of viable bacteria and for the histopathological changes. The numbers of bacteria were greater and histopathological changes were severer in these organs of mice inoculated with strain AL1190, than in those with strain AL1192. Strains AL1190, AL1192, and AL1193 showed the equivalent survival rate to sera of guinea pig, calf, and pig, and grew equally well under the iron-limiting conditions. These results suggested that the 36 Md plasmid of S. Enteritidis contribute to the spread of the infection beyond small intestines to mesenteric lymph nodes, spleens, and livers, but not to serum resistance or acquisition of iron.