Shigella and enteroinvasive Escherichia coli (EIEC) strains secrete virulence proteins by a complex machinery called the type III secretion (TTS) apparatus. Secretion of virulence proteins is a tightly-regulated phenomenon such that the TTS system is weakly active when bacteria are grown in common laboratory media. Activation of the TTS system is triggered by contact with eukaryotic cells, or can be artificially stimulated by the addition of Congo red dye to the growth medium. Exploiting the ability of bovine lactoferrin (bLf) to bind iron we have found that the TTS of EIEC strain HN280 seems to be activated in conditions of low-iron availability, obtained by incubation of bacteria with bLf enclosed within a dialysis bag. Activation of secretion was assessed by measuring the release of IpaB and C, chosen as reporters of secreted virulence proteins. The contribution of small bLf-derived components, diffusing across the dialysis membrane, in the release of Ipa proteins has also been determined. Activation of secretion was not due to bLf-induced damage of the HN280 outer membrane and was not associated with increased transcription of the mxi operon. Thus, low-iron availability might be an environmental signal perceived by enteroinvasive micro-organisms in order to modulate secretion of virulence proteins.