Using a combination of experimental studies, theory, simulation, and modeling, we investigate the hydrogen atom transfer (HAT) reaction by the high-valent ferryl cytochrome P450 (CYP) intermediate known as Compound I, a species that is central to innumerable and important detoxification and biosynthetic reactions. The P450 decarboxylase known as OleT converts fatty acids, a sustainable biological feedstock, into terminal alkenes and thus is of high interest as a potential means to produce fungible biofuels. Previous experimental work has established the intermediacy of Compound I in the C─C scission reaction catalyzed by OleT and an unprecedented ability to monitor the HAT process in the presence of bound fatty acid substrates. Here, we leverage the kinetic simplicity of the OleT system to measure the activation barriers for CYP HAT and the temperature dependence of the substrate 2H kinetic isotope effect. Notably, neither measurement has been previously accessible for a CYP to date. Theoretical analysis alludes to the significance of substrate fatty acid coordination for generating the hydrogen donor/acceptor configurations that are most conducive for HAT to occur. The analysis of the two-dimensional potential energy surface, based on multireference electronic wave functions, illustrates the uncoupled character of the hydrogen motion. Quantum dynamics calculations along the hydrogen reaction path demonstrate that hydrogen tunneling is essential to qualitatively capture the experimental isotope effect, its temperature dependence, and appropriate activation energies. Overall, a more fundamental understanding of the OleT reaction coordinate contributes to the development of biomimetic catalysts for controlled C─H bond activation, an outstanding current challenge for (bio)synthetic chemistry.