In iron-limiting environments, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium synthesize and secrete several types of siderophore to trap trivalent ferric ions; these bacteria then express siderophore receptors called iron-regulated outer membrane proteins (IROMPs). In this study, we experimentally reproduced iron-limiting environments using a divalent metal chelator. IroN, one of the IROMPs, was purified by affinity chromatography with an anti-IroN-MAb-immobilized column. Thirty-day-old chickens were immunized intramuscularly with purified IroN from Salmonella Typhimurium mixed with Freund's incomplete adjuvant; the chickens were then challenged intravenously with Salmonella Enteritidis. The mortality rate of immunized chickens was 10%. On the other hand, that of control chickens was 80%. By Western blot analysis, specific IgG antibody responses against IroN of Salmonella Enteritidis were identified in chickens immunized with purified IroN. These results indicate that IroN might be promising as an important vaccine component against Salmonella infection in chickens.