Atractylenolide III (AT-III) is a pharmacologically effective phytochemical and is known to be oxygenated during systemic metabolism mainly by cytochrome P450 enzymes (CYP450s), iron-containing porphyrin-based oxygenases. In rat plasma samples, the oxygenated metabolite of orally ingested AT-III was determined using liquid chromatography/mass spectrometry and the oxygenated form of AT-III was maintained at higher levels than the original form of AT-III. In situ catalytic reactions using the iron(IV)-oxo porphyrin π-cation radical complex, [(tmp⁺˙)Fe^IV(O)]⁺, demonstrated that both H-atom abstraction and an oxygen rebound mechanism participated in the oxygenation process of AT-III. Density functional theory (DFT) confirmed the oxidative transformation occurred at the 4th and 10th carbon positions of AT-III. Co-treatment with acetaminophen had different effects between in vivo and in situ models of AT-III metabolism. AT-III was metabolized via an oxygenation process in the rat body, where CYP450 and other O₂-activating metalloenzymes might participate in the metabolism. The present work provided the oxidative metabolism of AT-III using an in vivo model parallel with in situ biomimetic reaction models.