Oxygen therapy to maintain tissue oxygenation is one of the cornerstones of critical care. Therefore, hyperoxia is often encountered in critically ill patients. Epidemiologic studies have demonstrated that hyperoxia may affect outcome, although mechanisms are unclear. Immunologic effects might be involved, as hyperoxia was shown to attenuate inflammation and organ damage in preclinical models. However, it remains unclear whether these observations can be ascribed to direct immunosuppressive effects of hyperoxia or to preserved tissue oxygenation. In contrast to these putative anti-inflammatory effects, hyperoxia may elicit an inflammatory response and organ damage in itself, known as oxygen toxicity. Here, we demonstrate that, in the absence of systemic inflammation, short-term hyperoxia (100% O2 for 2.5 hours in mice and 3.5 hours in humans) does not result in increased levels of inflammatory cytokines in both mice and healthy volunteers. Furthermore, we show that, compared with room air, hyperoxia does not affect the systemic inflammatory response elicited by administration of bacterial endotoxin in mice and man. Finally, neutrophil phagocytosis and ROS generation are unaffected by short-term hyperoxia. Our results indicate that hyperoxia does not exert direct anti-inflammatory effects and temper expectations of using it as an immunomodulatory treatment strategy.