Plants produce flavonol compounds for vital functions regarding plant growth, fruit and flower colouring as well as fruit ripening processes. Several of these biosynthesis steps are stereo- and regioselective and are being carried out by nonheme iron enzymes. Using density functional theory calculations on a large active site model complex of flavanone-3β-hydroxylase (FHT), we established the mechanism for conversion of naringenin to its dihydroflavonol, which is a key step in the mechanism of flavonol biosynthesis. The reaction starts with dioxygen binding to the iron(II) centre and a reaction with α-ketoglutarate co-substrate gives succinate, an iron(IV)-oxo species and CO2 with large exothermicity and small reaction barriers. The rate-determining reaction step in the mechanism; however, is hydrogen atom abstraction of an aliphatic C‒H bond by the iron(IV)-oxo species. We identify a large kinetic isotope effect for the replacement of the transferring hydrogen atom by deuterium. In a final step the OH and substrate radicals combine to form the alcohol product with a barrier of several kcal mol–1. We show that the latter is the result of geometric constraints in the active site pocket. Furthermore, the calculations show that a weak tertiary C‒H bond is shielded from the iron(IV)-oxo species in the substrate binding position and therefore the enzyme is able to activate a stronger C‒H bond. As such, the flavanone-3β-hydroxylase enzyme reacts regioselectively with one specific C‒H bond of naringenin by avoiding activation of weaker bonds through tight substrate and oxidant positioning.