Mechanical ventilation causes ventilator-induced lung injury (VILI), and contributes to acute lung injury/acute respiratory distress syndrome (ALI/ARDS), a disease with high morbidity and mortality among critically ill patients. Carbon monoxide (CO) can confer lung protective effects during mechanical ventilation. This study investigates the time dependency of CO therapy with respect to lung protection in animals subjected to mechanical ventilation. For this purpose, mice were ventilated with a tidal volume of 12 ml/kg body weight for 6 h with air in the absence or presence of CO (250 parts per million). Histological analysis of lung tissue sections was used to determine alveolar wall thickening and the degree of lung damage by VILI score. Bronchoalveolar lavage fluid was analyzed for total cellular influx, neutrophil accumulation, and interleukin-1β release. As the main results, mechanical ventilation induced pulmonary edema, cytokine release, and neutrophil recruitment. In contrast, application of CO for 6 h prevented VILI. Although CO application for 3 h followed by 3-h air ventilation failed to prevent lung injury, a further reduction of CO application time to 1 h in this setting provided sufficient protection. Pre-treatment of animals with inhaled CO for 1 h before ventilation showed no beneficial effect. Delayed application of CO beginning at 3 or 5 h after initiation of ventilation, reduced lung damage, total cell influx, and neutrophil accumulation. In conclusion, administration of CO for 6 h protected against VILI. Identical protective effects were achieved by limiting the administration of CO to the first hour of ventilation. Pre-treatment with CO had no impact on VILI. In contrast, delayed application of CO led to anti-inflammatory effects with time-dependent reduction in tissue protection.