Inoculation of legume seed with rhizobia is an efficient and cost-effective means of distributing elite rhizobial strains to broad-acre crops and pastures. However, necessary drying steps after coating seed expose rhizobia to desiccation stress reducing survival and limiting potential nitrogen fixation by legumes. Rhizobial tolerance to desiccation varies with strain and with growth conditions prior to drying. Cells grown in peat generally survive desiccation better than cells grown in liquid broth. We aimed to identify peat-induced proteomic changes in rhizobia that may be linked to desiccation tolerance. Proteins expressed differentially after growth in peat extract when compared with a minimal defined medium were measured in four rhizobial strains. Proteins showing the greatest increase in abundance were those involved in amino acid and carbohydrate transport and metabolism. Proteins involved in posttranslational modification and cell defence mechanisms were also upregulated. Many of the proteins identified in this study have been previously linked to stress responses. In addition, analysis using nucleic acid stains SYTO9 and propidium iodide indicated that membranes had been compromised after growth in peat extract. We targeted the membrane repair protein PspA (Delta RL3579) which was upregulated in Rhizobium leguminosarum bv. viceae 3841 after growth in peat extract to validate whether the inability to repair membrane damage after growth in peat extract reduced desiccation tolerance. The Delta RL3579 mutant grown in peat extract had significantly lower survival under desiccation stress, whereas no difference in survival between wild-type and mutant strains was observed after growth in tryptone yeast (TY) or minimal medium (JMM) media. Staining mutant and wild-type strains with SYTO9 and propidium iodide indicated that membranes of the mutant were compromised after growth in peat extract and to a lesser extent in TY. This study shows that growth in peat extract causes damage to cell membranes and exposes rhizobia to sub-lethal stress resulting in differential expression of several stress-induced proteins. The induction of these proteins may prime and protect the cells when subjected to subsequent stress such as desiccation. Identifying the key proteins involved in desiccation tolerance and properties of peat that stimulate this response will be important to inform development of new inoculant technology that maximises survival of rhizobia during delivery to legume crops and pastures.