Crystallographic studies on flavodiiron proteins (FDPs) have revealed that the common sequence core (≈400 residues) that defines this protein family comprises two structural domains. The N-terminal domain (of approximately 250 residues) displays a metallo-β-lactamase-like-fold, being indeed structurally homologous to β-lactamases and glyoxalases, despite the poor sequence similarity. Whereas β-lactamases have mono- or dizinc sites and glyoxalases a mixed iron-zinc site, the lactamase domain of FDPs harbors a nonheme diiron center with carboxylate and histidine residues as ligands, assigned as the active site of NO and/or O2 reduction. The C-terminal domain of FDPs is characterized by a flavodoxin-like fold, homologous to short-chain flavodoxins, and harbors a flavin mononucleotide moiety, stabilized by van der Waals interactions and a number of hydrogen bonds. Structures of FDPs obtained in different conditions and oxidation states display some heterogeneities, mostly at the diiron site, but still fail to provide unequivocal evidence for some pending questions regarding the substrate activation mechanism of FDPs, namely the preference for either substrate (NO or oxygen) observed in different members of this protein family. More structural studies are therefore required to achieve a deeper understanding on these matters.