NtdB is an enzyme encoded within the ntd operon in Bacillus subtilis. This operon is reported to contain a complete set of genes necessary for the biosynthesis of 3,3'-neotrehalosadiamine (NTD), a compound composed of two kanosamine subunits linked together by a 1,1'-(α,β)-linkage. Both NTD and kanosamine have reported antibiotic properties. The function of NtdB has been a matter of speculation, but has never been investigated in vitro. Using a phosphate assay and an array of substrates, NtdB was determined to be a phosphatase, specific to kanosamine-6-phosphate (K6P) (kcat = 32 ± 1 s-1, Km = 93 ± 7 µM). Site-directed mutagenesis of amino acid residues in the core and the cap domains of the enzyme identified residues important for the catalytic reaction and substrate specificity. These mutations confirmed the presence of four motifs, characteristic of members of the haloacid dehalogenase (HAD) superfamily, and allowed identification of the substrate binding site of the enzyme. KabB, a homologue of NtdB from Bacillus cereus, showed notably lower activity with K6P than NtdB. This research defines the role of NtdB as a specific K6P phosphatase and challenges the previously reported NTD biosynthesis pathway by demonstrating a novel pathway for the production of the antibiotic kanosamine.