The pathogenesis of ventilator-induced lung injury has predominantly been attributed to overdistension or mechanical opening and collapse of alveoli, while mechanical strain on the airways is rarely taken into consideration. Here, we hypothesized that mechanical ventilation may cause significant airway distension which may contribute to the pathologic features of ventilator-induced lung injury. C57BL/6J mice were anesthetized and mechanically ventilated at tidal volumes of 6, 10 or 15 mL/kg body weight. Mice were imaged by flat-panel volume computer tomography, central airways were segmented and rendered in 3D for quantitative assessment of airway distension. Alveolar distension was imaged by intravital microscopy. Functional dead space was analyzed in vivo, and pro-inflammatory cytokine release in isolated, ventilated tracheae. CT scans revealed a reversible, up to 2.5-fold increase in upper airway volume during mechanical ventilation as compared to spontaneous breathing. Airway distension was most pronounced in main bronchi which showed the largest volumes at tidal volumes of 10 mL/kg body weight. Conversely, airway distension in segmental bronchi and functional dead space increased almost linearly, and alveolar distension even disproportionately with higher tidal volumes. In isolated tracheae, mechanical ventilation stimulated the release of the early-response cytokines TNF-α and IL-1β. Mechanical ventilation causes a rapid, pronounced and reversible distension of upper airways in mice that is associated with an increase in functional dead space. Upper airway distension is most pronounced at moderate tidal volumes, while higher tidal volumes redistribute preferentially to the alveolar compartment. Airway distension triggers pro-inflammatory responses, and may thus contribute relevantly to ventilator-induced pathologies.