The last step in the biosynthetic pathway of the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is catalyzed by Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase (FaQR). The ripening-induced enzyme catalyzes the reduction of the exocyclic double bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) in a NAD(P)H dependent manner. To elucidate the molecular mechanism of this peculiar reaction, we determined the crystal structure of FaEO in altogether six different states or complexes at resolutions of ≤ 1.6 Å, including those with HDMF as well as with three substrate analogs. Our crystallographic analysis revealed a monomeric enzyme whose active site is largely determined by the bound NAD(P)H cofactor, which is embedded into a Rossmann fold. Considering that the quasi-symmetric enolic reaction product HDMF is prone to extensive tautomerization, whereas its precursor HMMF is chemically labile in aqueous solution, we used the asymmetric and more stable surrogate product 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone (EHMF) and the corresponding substrate (2E)-ethylidene-4-hydroxy-5-methyl-3(2H)-furanone (EDHMF) to study their enzyme complexes as well. Together with deuterium-labeling experiments of EDHMF with [4R-2H]-NADH and chiral phase analyses of the reaction product EHMF, our data reveal that the 4R-hydride of NAD(P)H is transferred to the unsaturated exocyclic C6 carbon of HMMF, resulting in a cyclic non-chiral enolate intermediate that subsequently becomes protonated, eventually leading to HDMF. Apart from elucidating this important reaction of the secondary metabolism in plant fruit our study provides a foundation for protein engineering of enone oxidoreductases and their application in biocatalytic processes.