3,3'-Neotrehalosadiamine (NTD) is a diaminosugar that possesses a rare alpha,beta-1,1'-linked glycosidic bond and has been reported to possess antimicrobial activity against Staphylococcus aureus. The ntdABC operon contains three structural genes that are necessary for the production of NTD in certain mutants of Bacillus subtilis. The gene predicted to be the first in the NTD biosynthetic pathway, ntdC, was subcloned into pET-28b as the hexa-histidine tagged fusion. The gene product was expressed, purified to homogeneity, and found to be an NAD+-dependent glucose 6-phosphate 3-dehydrogenase, likely operating according to a ternary complex mechanism and possessing a catalytic dyad composed by D176 and H180. The advent of this knowledge suggests that additional genes are required for the biosynthesis of NTD aside from the three encoded by the ntdABC operon.