The plant non-heme iron dioxygenase flavonol synthase performs a regioselective desaturation reaction as part of the biosynthesis of the signaling molecule flavonol that triggers the growing of leaves and flowers. These compounds also have health benefits for humans. Desaturation of aliphatic compounds generally proceeds through two consecutive hydrogen atom abstraction steps from two adjacent carbon atoms and in nature often is performed by a high-valent iron(IV)-oxo species. We show that the order of the hydrogen atom abstraction steps, however, is opposite of those expected from the C-H bond strengths in the substrate and determines the product distributions. As such, flavonol synthase follows a negative catalysis mechanism. Using density functional theory methods on large active-site model complexes, we investigated pathways for desaturation and hydroxylation by an iron(IV)-oxo active-site model. Contrary to thermochemical predictions, we find that the oxidant abstracts the hydrogen atom from the strong C-2-H bond rather than the weaker C-3-H bond of the substrate first. We analyze the origin of this unexpected selective hydrogen atom abstraction pathway and find that the alternative C-3-H hydrogen atom abstraction would be followed by a low-energy and competitive substrate hydroxylation mechanism hence, should give considerable amount of byproducts. Our computational modeling studies show that substrate positioning in flavonol synthase is essential, as it guides the reactivity to a chemo- and regioselective substrate desaturation from the C-2-H group, leading to desaturation products efficiently.