The crystal structure of Bacillus subtilis RibG at 2.41-Å resolution displayed a tetrameric ring-like structure with an extensive interface of ~2400 Å2 per monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation, but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities, allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains, and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.