Propionibacterium freudenreichii is a beneficial bacterium used as a cheese starter and as a probiotic. Indeed,selected strains of P. freudenreichii combine both technological and health-promoting abilities. Moreover, duringlarge-scale industrial production of dried bacteria and during consumption, P. freudenreichii may undergo differentstressful processes. Osmotic adaptation was shown to enhance P. freudenreichii tolerance towards stresses,which are encountered during freeze-drying and during digestion. In this report, we compared the osmoadaptationmolecular mechanisms of two P. freudenreichii strains. Both osmotolerance and osmoadaptation werestrain-dependent and had different effects on multiple stress tolerance, depending on the presence of osmoprotectants.Availability of glycine betaine (GB) restored the growth of one of the two strains. In this strain,osmotic preadaptation enhanced heat, oxidative and acid stresses tolerance, as well as survival upon freezedrying.However, addition of GB in the medium had deleterious effects on stress tolerance, while restoringoptimal growth under hyperosmotic constraint. In the other strain, neither salt nor GB enhanced stress tolerance,which was constitutively low. Accordingly, whole cell proteomics revealed that mechanisms triggered by salt inthe presence and in the absence of GB are different between strains. Osmotic adjustment may thus have deleteriouseffects on industrial abilities of P. freudenreichii.Biological significance: Propionibacteria are found in various niches including fodder, silage, rumen, milk andcheeses. This means adaptation towards different ecological environments with different physicochemicalparameters. Propionibacterium freudenreichii, in particular, is furthermore used both as dairy starter and asprobiotic and is thus submitted to high scale industrial production. Production and subsequent stabilization stillneed optimization. Drying processes like freeze-drying are stressful. Osmotic adjustments may modulated tolerancetowards drying. However, they are strain-dependent, medium-dependent and may either reduce or increasestress tolerance. A case-by-case study, for each strain-medium thus seems necessary. In this work, weidentify key proteins involved in osmoadaptation and give new insights into adaptation mechanisms in P.freudenreichii. This opens new perspectives for the selections of strains and for the choice of the growth medium composition.