Cellular hypoxia plays a crucial role in tissue development and adaptation to pO2. Central to cellular oxygen sensing is factor-inhibiting HIF-1α (FIH), an α-ketoglutarate (αKG)/non-heme iron(II)-dependent dioxygenase that hydroxylates a specific asparagine residue of hypoxia inducible factor-1α (HIF-1α). The high KM(O2) and rate-limiting decarboxylation step upon O2 activation are key features of the enzyme that classify it as an oxygen sensor and set it apart from other αKG/Fe(II)-dependent dioxygenases. Although the chemical intermediates following decarboxylation are presumed to follow the consensus mechanism of other αKG/Fe(II)-dependent dioxygenases, experiments have not previously demonstrated these canonical steps in FIH. In this work, a deuterated peptide substrate was used as a mechanistic probe for the canonical hydrogen atom transfer (HAT). Our data show a large kinetic isotope effect (KIE) in steady-state kinetics (Dkcat = 10 ± 1), revealing that the HAT occurs and is partially rate limiting on kcat. Kinetic studies showed that the deuterated peptide led FIH to uncouple O2 activation and provided the opportunity to spectroscopically observe the ferryl intermediate. This enzyme uncoupling was used as an internal competition with respect to the fate of the ferryl intermediate, demonstrating a large observed KIE on the uncoupling (Dk5 = 1.147 ± 0.005) and an intrinsic KIE on the HAT step (Dk > 15). The close energy barrier between αKG decarboxylation and HAT distinguishes FIH as an O2-sensing enzyme and is crucial for ensuring substrate specificity in the regulation of cellular O2 homeostasis.