ABSTRACTVancomycin-resistant enterococci (VRE) have become a major nosocomial threat.Enterococcus faeciumis of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studiedLactobacillus rhamnosusstrain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates andL. rhamnosusGG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster ofL. rhamnosusGG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili ofL. rhamnosusGG and those of the clinicalE. faeciumstrain E1165, which was characterized at the genome level. Moreover,E. faeciumstrain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies againstL. rhamnosusGG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili ofL. rhamnosusGG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation ofL. rhamnosusGG and its pilins for prophylaxis and treatment of VRE infections.IMPORTANCEConcern about vancomycin-resistantEnterococcus faeciumcausing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application ofLactobacillus rhamnosusGG in VRE decolonization strategies. Our results provide support for a new molecular mechanism, in which probiotics can perform competitive exclusion and possibly immune interaction. Moreover, we spur further exploration of the potential of intactL. rhamnosusGG and purified SpaC pilin as prophylactic and curative agents of the VRE carrier state.