We have previously shown that streptozotocin (STZ) inhibits O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from proteins. In light of this observation, we explored the possibility that the diabetogenic toxin alloxan, an O-GlcNAc transferase (OGT) inhibitor, might also inhibit O-GlcNAcase. Alloxan inhibited islet O-GlcNAcase with a dose-response much like that of STZ. Similar to STZ, islet O-GlcNAcase was more susceptible to alloxan inhibition than was brain O-GlcNAcase. Alloxan directly inhibited recombinant O-GlcNAcase activity with a dose-response very similar to that of STZ. Subsequent LC/MS/MS analysis revealed that alloxan modified the tryptic digest pattern of the enzyme. One tryptic peptide LGCFEIAK(894-901) was modified by alloxan. Two other tryptic peptides, LDQVSQFGCR(158-167) and SFALLFDDIDHNMCAADK(168-185), both N-terminal active site peptides, were absent after alloxan treatment. Together, these data demonstrate that alloxan is an inhibitor of O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase, with inhibition corresponding to an altered tryptic digest pattern of N-terminal active site peptides.