The ntd operon in Bacillus subtilis is essential for biosynthesis of 3,3'-neotrehalosadiamine (NTD), an unusual nonreducing disaccharide reported to have antibiotic properties. It has been proposed that the three enzymes encoded within this operon, NtdA, NtdB, and NtdC, constitute a complete set of enzymes required for NTD synthesis, although their functions have never been demonstrated in vitro. We now report that these enzymes catalyze the biosynthesis of kanosamine from glucose-6-phosphate: NtdC is a glucose-6-phosphate 3-dehydrogenase, NtdA is a pyridoxal phosphate-dependent 3-oxo-glucose-6-phosphate:glutamate aminotransferase, and NtdB is a kanosamine-6-phosphate phosphatase. None of these enzymatic reactions have been reported before. This pathway represents an alternate route to the previously reported pathway from Amycolatopsis mediterranei which derives kanosamine from UDP-glucose.