The purpose of this study was to measure the regional change of magnetic susceptibility in human brain upon inhalation of 100% oxygen by MRI quantitative susceptibility mapping (QSM). Fourteen healthy volunteers were scanned in a 3 T MR scanner with a 3D multi‐gradient‐echo sequence while breathing medical air (normoxia) and pure oxygen (hyperoxia). QSM images and R2* maps were calculated. Mean susceptibility differences versus white matter were measured in regions of interest covering veins, gray matter (GM), and cerebrospinal fluid (CSF) under both conditions. Hyperoxia resulted in a strong susceptibility decrease in large veins (−154.4 ± 65.9 ppb, p < 10−6), in a smaller reduction in GM (−1.3 ± 1 ppb, p < 0.001), and in a susceptibility increase in ventricular CSF (3.8 ± 1.8 ppb, p < 10−5). The susceptibility decrease in veins implied an increase of venous oxygen saturation (SvO2) by 10.1 ± 4.0%. Compared with QSM, R2* was more seriously affected by long‐distance effects not related to local tissue oxygenation and increased in cerebral frontal regions (3 ± 2 s−1, p < 0.0004) due to paramagnetic molecular oxygen in cavities. The results highlight the potential of QSM to yield region‐specific quantitative oxygenation information, and, thus, for applications such as oxygen‐therapy monitoring or identification of hypoxic tumor tissue during radiotherapy planning. Copyright © 2015 John Wiley & Sons, Ltd.