A metabolic pathway for biosynthesis of the nonreducing disaccharide mannosylfructose (beta-fructofuranosyl-alpha-mannopyranoside), an important osmolyte in Agrobacterium tumefaciens, was discovered. We have identified and functionally characterized two ORFs that correspond to genes (named mfpsA and mfppA) encoding the rare enzymes mannosylfructose-phosphate synthase and mannosylfructose-phosphate phosphatase, an associated phosphohydrolase. The mfpsA and mfppA genes are arranged in an operon structure, whose transcription is up-regulated by NaCl, resulting in the accumulation of mannosylfructose in the cells. Not only is the biosynthesis of mannosylfructose mechanistically similar to that of sucrose, but the corresponding genes for the biosynthesis of both disaccharides are also phylogenetic close relatives. Importantly, a protein phylogeny analysis indicated that mannosylfructose-phosphate synthase defines a unique group of mannosyltransferases.