Introduction. Pseudomonas aeruginosa (PsA) ventilator-associated pneumonia (VAP) is a major cause of nosocomial infections in intensive care units and is associated with increased morbidity and mortality. Mechanical ventilation (MV), although a life-saving intervention, it may also exacerbate pre-existing lung injury, a process termed ventilator-induced lung injury (VILI). Whether MV and the associated inflammation have an impact on the development of VAP remains majorly unknown. Methods. We established a new murine model of VAP to explore the influence of MV on the development of PsA pneumonia. Sedated C57Bl/6J female mice were subjected to MV. VILI was induced by high tidal volume ventilation (HVt: 34ml/kg). Protectively ventilated (LVt; low Vt: 9ml/kg) animals were used as control. After 4h, mice were detached from the ventilator and PsA was instilled via the ventilation tube. PBS treated animals were used as sham-infected controls. After infection, sedation was antagonized, and the animals were extubated and breathed spontaneously for 24h. Respiratory function was tested at start and end of MV. Lung permeability, inflammatory mediators, recruited inflammatory cells, and bacterial load (CFU -colony forming units) in lung, blood, liver, and spleen were assessed 24h post infection. Results. HVt-MV led to an increased mean airway pressure and decreased lung compliance after 4h of MV (vs LVt). In sham-infected mice (24h after MV), despite a 3-fold increase in pulmonary permeability and 2-fold increase in IL-6 levels in the HVt group (vs LVt), there were no significant differences in inflammatory cell recruitment or the levels of other inflammatory mediators (MCP-1, CXCL1, IL1-β). Infection with PsA led to higher levels of inflammation. After HVt-MV, PsA-infected mice had significantly increased alveolar-capillary permeability (10-fold), increased protein and gene expression of inflammatory mediators (IL-6, MCP-1, CXCL1, IL1-β), and enhanced neutrophil recruitment to the lungs compared to LVt-PsA infected mice. In HVt-PsA group, lung tissue bacteria counts were significantly higher than in LVt-PsA mice. Interestingly, extra pulmonary spread of bacteria (blood, spleen, liver) and signs of systemic inflammation (increased TNF-α, MCP-1 and neutrophil counts) were only observed in HVt-PsA group. Conclusion. Mice subjected to HVt-MV (VILI) were more susceptible to PsA infection and developed sepsis, whereas protectively (LVt) ventilated animals only developed a mild local pneumonia. This novel murine PsA VAP model shows that MV-associated inflammation contributes to the development of pneumonia and may thus enable for mechanistic investigations on the pathophysiology of VAP.