Random transposon mutagenesis led to the isolation of a novel Mesorhizobium loti mutant that is defective in nitrogen fixation during symbiosis with Lotus japonicus. The mutated locus, designated cep, encodes a putative cell-envelope protein displaying no significant sequence similarity to proteins with known functions. This mutant elicits the formation of nodule-like bumps and root-hair curling, but not the elongation of infection threads, on L. japonicus roots. This is reminiscent of the phenotypes of rhizobial mutants impaired in cyclic β-glucan biosynthesis. The cep mutant exhibits partially reduced content of cell-associated glucans and intermediate deficiency of motility under hypo-osmotic conditions as compared to a glucan-deficient mutant. Second-site pseudorevertants of the cep mutant were isolated by selecting for restoration of symbiotic nitrogen fixation. A subset of pseudorevertants restored both symbiotic capability and glucan content to levels comparable to that of the wild-type. These results suggest that the Cep product acts on a successful symbiosis by affecting cell-associated glucan content.