Prodigiosin is an intensely red pigment comprised of three pyrroles. The biosynthetic pathway includes a two-step proline oxidation catalyzed by PigA, using FAD as its cofactor. Here the enzyme is crystallized in apo and FAD-bound forms. The protein folds into two α-helical domains (I and III) separated by a β-sheet domain (II) and it belongs to the acyl-CoA dehydrogenase (ACAD) family. In the tetrameric enzyme, which comprises two dimers associated via domain III, FAD is located in a cleft surrounded by all three domains of one monomer and domain III of another. The overall structure as well as the FAD-binding mode is similar to those of other ACAD-family enzymes. Alternate backbone conformations in the N-terminal part of helix αG correlate well with the expected location of substrate to the Re side of FAD. Complex modeling with PigG, the acyl carrier protein, suggests a plausible binding mode. The structure helps explain the proline oxidation mechanism, in which Glu244 plays a central role in the double-bond formations. It also reveals a plausible pocket for oxygen binding and reduction on the Si side of FAD.