Given the increasing commercial and clinical relevance of probiotic cultures, improving their stress tolerance profile and ability to overcome the physiological defences of the host is an important biological goal. In order to reach the gastrointestinal tract in sufficient numbers to exert a therapeutic effect, probiotic bacteria must resist the deleterious actions of low pH, elevated osmolarity and bile salts. Cloning the listerial betaine uptake system, BetL, into the probiotic strain Bifidobacterium breve UCC2003 significantly improved probiotic tolerance to gastric juice and conditions of elevated osmolarity mimicking the gut environment. Furthermore, whilst stable colonization of the murine intestine was achieved by oral administration of B. breve UCC2003, strains harbouring BetL were recovered at significantly higher levels in the faeces, intestines and caecum of inoculated animals. Finally, in addition to improved gastric transit and intestinal persistence, this approach improved the clinical efficacy of the probiotic culture: mice fed B. breve UCC2003-BetL(+) exhibited significantly lower levels of systemic infection compared to the control strain following oral inoculation with Listeria monocytogenes.