Iron is a growth requirement for virtually all microbes. In the human body, extracellular iron is sequestered from microbes by binding to proteins such as lactoferrin. In this study the effect of lactoferrin and human milk on Yersinia pseudotuberculosis was investigated. Its growth in vitro was inhibited by iron-free, but not iron saturated, pure lactoferrin or human milk. Iron-free human milk and to a less extent normal human milk were bactericidal for Y. pseudotuberculosis cells that were suspended in deionized water. The in vivo studies also show that iron-saturated lactoferrin enhanced growth, whereas, the viable count was reduced by iron-free pure lactoferrin and EDDA. Nine envelope proteins were decreased or disappeared upon growth in iron-deficient medium, whereas one new high molecular weight protein appeared under the same conditions.