Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) is a key enzyme of the purine-salvage pathway. Its ability to transfer glycosyl residues to acceptor bases is of great biotechnological interest owing to its potential application in the synthesis of nucleoside analogues used in the treatment of antiviral infections and in anticancer chemotherapy. Although hexameric PNPs are prevalent in prokaryotes, some microorganisms, such as Bacillus subtilis, present both hexameric and trimeric PNPs. The hexameric PNP from B. subtilis strain 168, named BsPNP233, was cloned, expressed and crystallized. Crystals belonging to different space groups (P32(1), P2(1)2(1)2(1), P6(3)22 and H32) were grown in distinct conditions with pH values ranging from 4.2 to 10.5. The crystals diffracted to maximum resolutions ranging from 2.65 to 1.70 Å.