Biocatalytic reduction of α- or β-alkyl-β-arylnitroalkenes provides a convenient and efficient method to prepare chiral substituted nitroalkanes. Pentaerythritol tetranitrate reductase (PETN reductase) from Enterobacter cloacae st. PB2 catalyses the reduction of nitroolefins such as 1-nitrocyclohexene ( 1 ) with steady state and rapid reaction kinetics comparable to other old yellow enzyme homologues. Furthermore, it reduces 2-aryl-1-nitropropenes ( 4a-d ) to their equivalent ( S )-nitropropanes 9a-d . The enzyme shows a preference for the ( Z )-isomer of substrates 4a-d , providing almost pure enantiomeric products 9a-d ( ee s up to > 99%) in quantitative yield, whereas the respective ( E )-isomers are reduced with lower enantioselectivity (63-89% ee ) and lower product yields. 1-Aryl-2-nitropropenes ( 5a , b ) are also reduced efficiently, but the products ( R )- 10 have lower optical purities. The structure of the enzyme complex with 1-nitrocyclohexene ( 1 ) was determined by X-ray crystallography, revealing two substrate-binding modes, with only one compatible with hydride transfer. Models of nitropropenes 4 and 5 in the active site of PETN reductase predicted that the enantioselectivity of the reaction was dependent on the orientation of binding of the ( E )- and ( Z )-substrates. This work provides a structural basis for understanding the mechanism of asymmetric bioreduction of nitroalkenes by PETN reductase.