Spores of Bacillus subtilis are covered by a multi-protein protective coat which is a key factor in their extreme environmental resilience. A fraction of the coat proteins undergoes covalent cross-linking following their assembly at the spore surface. Several types of covalent cross-links are found in the coat. These include epsilon-(gamma-glutamyl)lysine bonds whose formation is catalyzed by a transglutaminase, Tgl, itself a coat component. Tgl is the smallest known transglutaminase. It bears no sequence resemblance to other proteins in databases, except for its counterparts in other Bacillus and related species, suggesting a highly specialized role in coat assembly. It is not known to what degree are the Tgl-like proteins structural and mechanistically related to other transglutaminases. Here, we have fused the His(6) tag to the C-terminal end of Tgl, and shown that the fusion protein is functional in vivo. We have overproduced B. subtilis Tgl-His(6) by auto-induction with high yield and purified the protein to nearly homogeneity in a single chromatographic step. The purified protein, active as it catalyzed the cross-linking of bovine serum albumin, behaved as a monomer of about 33kDa in solution. Lastly, Tgl was crystallized and X-ray diffraction data were collected using synchrotron radiation to 2.1A resolution. Crystals of Tgl belong to the tetragonal space group P4(1,3) and contain two molecules per asymmetric unit.