Rhizobium leguminosarum is a plant-associated bacterium that can form a symbiotic relationship with leguminous plants. Rhizobia must respond to significantly different environments during their biphasic lifestyle. The cell envelope is an important cellular feature that must be able to adapt to changing environments. Mutations in rhizobial genes required for proper cell envelope development have been identified based on growth deficiencies on peptide-rich media. Using transposon mutagenesis and screening of mutants for loss of growth on peptide-rich media, this study identified RL4716 as being required for proper cell envelope function in R. leguminosarum. Mutation of RL4716 results in an altered cell morphology, and an increase in permeability to the non-polar probe 1-N-phenylnaphthylamine, indicating a role of RL4716 in maintaining cell envelope integrity. The mutation also affected phenotypes that are known to be dependent on genes associated with a functional cell envelope including decreased desiccation tolerance and a decreased ability to form biofilms.