Vanadyl species are frequently employed as structural mimics of the fleeting Fe(IV)=O intermediate in C–H hydroxylation carried out by non-heme iron hydroxylases. We study active site models of non-heme iron hydroxylases and their vanadium-based mimics using density functional theory to determine if vanadyl is a faithful structural mimic. We identify crucial structural and energetic differences between ferryl and vanadyl isomers owing to the differences in their ground electronic states, i.e., high-spin (HS) for Fe and low-spin (LS) for V. For the succinate cofactor bound to the ferryl intermediate, we predict facile interconversion between monodentate and bidentate coordination isomers for ferryl species but difficult rearrangement for vanadyl mimics. We study isomerization of the oxo intermediate between axial and equatorial positions and find the ferryl potential energy surface to be characterized by a large barrier of ca. 10 kcal/mol that is completely absent for the vanadyl mimic. This analysis reveals even starker contrasts between Fe and V in hydroxylases than have been observed for this metal substitution in non-heme halogenases. Analysis of the relative bond strengths of coordinating carboxylate ligands for Fe and V reveals that all the ligands show stronger binding to V than Fe owing to the LS ground state of V in contrast to the HS ground state of Fe. Overall, the differences in structures, isomer energies, and isomerization energy landscapes between Fe and V highlight the limitations of vanadyl mimics of native non-heme iron hydroxylases.