LF82, a prototype of adherent-invasive (AIEC), is able to adhere to, invade, survive and replicate into intestinal epithelial cells. LF82 is able to enhance either its adhesion and invasion by up-regulating carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM-6), the main cell surface molecule for bacterial adhesion, and its intracellular survival by inducing host DNA damage, thus blocking the cellular cycle. Lactoferrin (Lf) is a multifunctional cationic glycoprotein of natural immunity, exerting an anti-invasive activity against LF82 when added to Caco-2 cells at the moment of infection. Here, the infection of 12 h Lf pre-treated Caco-2 cells was carried out at a time of 0 or 3 or 10 h after Lf removal from culture medium. The effect of Lf pre-treatment on LF82 invasiveness, survival, cell DNA damage, CEACAM-6 expression, apoptosis induction, as well as on Lf subcellular localization, has been evaluated. Lf, even if removed from culture medium, reduced LF82 invasion and survival as well as bacteria-induced DNA damage in Caco-2 cells independently from induction of apoptosis, modulation of CEACAM-6 expression and Lf sub-cellular localization. At our knowledge, this is the first study showing that the sole Lf pre-treatment can activate protective intracellular pathways, reducing LF82 invasiveness, intracellular survival and cell-DNA damages.