Allicin is a component of the characteristic smell and flavor of garlic (Allium sativum). A flavin-containing monooxygenase (FMO) produced by A. sativum (AsFMO) was previously proposed to oxidize S-allyl-L-cysteine (SAC) to alliin, an allicin precursor. Here, we present a kinetic and structural characterization of AsFMO that suggests a possible contradiction to this proposal. Results of steady-state kinetic analyses revealed that AsFMO exhibits negligible activity with SAC; however, the enzyme was highly active with L-cysteine, N-acetyl-L-cysteine, and allyl mercaptan. We found that allyl mercaptan with NADPH is the preferred substrate–cofactor combination. Rapid-reaction kinetic analyses showed that NADPH binds tightly (KD ~2 μM) to AsFMO and that the hydride transfer occurs with pro-R stereospecificity. We detected formation of a long-wavelength band when AsFMO was reduced by NADPH, probably representing the formation of a charge transfer complex. In the absence of substrate, the reduced enzyme, in complex with NADP+, reacted with oxygen and formed an intermediate with a spectrum characteristic of C4a-hydroperoxyflavin, which decays several orders of magnitude slower than the kcat. The presence of substrate enhanced C4a-hydroperoxyflavin formation, and upon hydroxylation, oxidation occurred at a rate constant similar to the kcat. The structure of AsFMO complexed with FAD at 2.08 A resolution features two domains for binding of FAD and NADPH, representative of class B flavin monooxygenases. These biochemical and structural results are consistent with AsFMO being an S-monooxygenase involved in allicin biosynthesis by direct formation of sulfenic acid, and not by SAC oxidation.