Vitamin B12 (cobalamin) is an essential micronutrient for humans that is synthesized by only a subset of bacteria and archaea. The aerobic biosynthesis of 5,6-dimethylbenzimidazole, the lower axial ligand of cobalamin, is catalyzed by the "flavin destructase" enzyme BluB, which fragments reduced flavin mononucleotide following its reaction with oxygen to yield this ligand. BluB is similar in sequence and structure to members of the flavin oxidoreductase superfamily, yet the flavin destruction process has remained elusive. Using stopped-flow spectrophotometry, we find that the flavin destructase reaction of BluB from Sinorhizobium meliloti is initiated with canonical flavin-O2 chemistry. A C4a-peroxyflavin intermediate is rapidly formed in BluB upon reaction with O2, and has properties similar to those of flavin-dependent hydroxylases. Analysis of reaction mixtures containing flavin analogues indicates that both formation of the C4a-peroxyflavin and the subsequent destruction of the flavin to form 5,6-dimethylbenzimidazole are influenced by the electronic properties of the flavin isoalloxazine ring. The flavin destruction phase of the reaction, which results from the decay of the C4a-peroxyflavin intermediate, occurs more efficiently at pH >7.5. Furthermore, the BluB mutants D32N and S167G are specifically impaired in the flavin destruction phase of the reaction; nevertheless, both form the C4a-peroxyflavin nearly quantitatively. Coupled with a phylogenetic analysis of BluB and related flavin-dependent enzymes, these results demonstrate that the BluB flavin destructase family can be identified by the presence of active site residues D32 and S167.