Aims: To assess the contribution of proline biosynthesis to listerial barotolerance. Methods and Results: Using a Listeria monocytogenes proBA deletion mutant, incapable of synthesizing proline, together with a proline-overproducing strain, the contribution of proline synthesis to listerial barotolerance was determined. The Delta proBA strain does not survive as well as the wild type when subjected to treatment of 500 MPa in rich media and 400 MPa in minimal media (c. 1 log lower survival in both conditions). Betaine and carnitine decrease the ability of the wild type to survive at low pressures (300 MPa), but confer normal or slightly increased levels of protection at higher pressures (350 and 400 MPa). Conclusions: A functional proline synthesis system is required for optimal survival of Listeria following treatment at high-pressure (HP) levels (500 MPa in brain heart infusion and 400 MPa in defined medium), particularly where other compatible solutes are absent or limiting. Significance and Impact of the Study: Given the potential of HP processing as an effective food processing/safety strategy, understanding how pathogens such as Listeria have evolved to cope with such stresses is an important food safety consideration. In this context, the work presented here may help to develop safer and more effective processing regimens.