Sepsis is a leading cause of death in patients with critical illness. However, there is no effective therapy available. Recent studies have suggested that ventilation with 100% oxygen (Oxy) can improve the survival rate and organ function in several shock models. However, a lot of work is necessary to be done before its clinical application, and its detailed mechanism remains to be clarified. In the present study, we showed that 100% Oxy inhalation for 2 or 3 h starting at 4 and 12 h after zymosan (ZY) injection, respectively, prevented the abnormal changes of serum biochemical parameters, tissue oxygenation, and organ histopathology, and improved the 14-day survival rate from 10% to 60% to 80% in mice. However, 100% Oxy inhalation for 1 or 4 h starting at the same time points has little preventive effects. We also showed that twice 100% Oxy inhalation for 2 or 3 h attenuated the increase of inflammatory cytokines and precluded the downregulation of antioxidant enzymatic activities. We further showed that the therapeutic time window of Oxy treatment against sterile sepsis was less than 12 h after ZY injection. We conclude that 100% Oxy inhalation for 2 or 3 h starting 4 and 12 h after ZY injection, respectively, protects against ZY-induced sterile sepsis via regulating inflammatory cytokines and antioxidant system in mice. The present results may provide a potential cue for developing more effective therapeutic strategies for patients with sepsis.