This study compared the immunomodulating properties of viable and killed Yersinia enterocolitica O9 in BALB/c mice. At 10 days after infection by the intragastric route, ex vivo assays showed a suppression of spleen cell proliferation in response to Salmonella lipopolysaccharide, concanavalin A and heat-killed yersiniae. Mice infected with Y. enterocolitica O9 for 10 days resisted the challenge with a lethal dose of Listeria monocytogenes. In contrast, intravenous administration of heat-killed yersiniae did not modify the ability of spleen cells to proliferate in response to lipopolysaccharide or concanavalin A, and proliferation in response to killed yersiniae was significantly increased. By 3 days after administration of a single dose of heat-killed yersiniae, the resistance of mice to L. monocytogenes challenge was significantly increased. Our findings show profound differences in immunomodulation by viable and heat-killed yersiniae, but suggest that killed yersiniae retain interesting immunomodulating properties.