Aims: To establish the relative importance of the osmo- and cryoprotective compounds glycine betaine and carnitine, and their transporters, for listerial growth and survival, in foods and during infection. Methods and Results: A set of Listeria monocytogenes mutants with single, double and triple mutations in the genes encoding the principal betaine and carnitine uptake systems (gbu, betL and opuC, respectively) was used to determine the specific contribution of each transporter to listerial growth and survival. Food models were chosen to represent high-risk foods of plant and animal origin i.e. coleslaw and frankfurters, which have previously been linked to major human outbreaks of listeriosis. BALB/c mice were used as an in vivo model of infection. Interestingly, while betaine appeared to confer most protection in foods, the hierarchy of transporter importance differs depending on the food type: Gbu>BetL>OpuC for coleslaw, as opposed to Gbu>OpuC>BetL in frankfurters. By contrast in the animal model, OpuC and thus carnitine, appears to play the dominant role, with the remaining systems contributing little to the infection process. Conclusions: This study demonstrates that the individual contribution of each system appears dependent on the immediate environment. In foods Gbu appears to play the dominant role, while during infection OpuC is most important. Significance and Impact of the Study: It is envisaged that this information may ultimately facilitate the design of effective control measures specifically targeting this pathogen in foods and during infection.