Mechanical ventilation (MV) is an essential life-support technique, but it can induce ventilator-induced lung injury (VILI) and subsequent pulmonary fibrosis. The mechanisms underlying this fibrosis are largely unknown. Because excessive polarization of M2 macrophages has increasingly been cited as possible inciting factor for tissue remodeling and organ fibrosis, we here hypothesize it might be involved in the development of pulmonary fibrosis after high tidal volume (VT) MV. In our prospective, randomized, controlled animal study, C57BL/6 mice were randomly placed in either a VILI group or sham group. After ventilation, surviving mice were allowed to recover for 0, 1, 3, 5, 7, or 14 days. 200 mice were involved in our in vivo experiment, and the results calculated here refer only to the surviving mice. The results clearly showed that high-VT MV caused early inflammation and a subsequent fibroproliferative response in mice without pre-existing lung disease. High-VT MV was also found to lead to a dramatic increase in the number of M2 macrophages in mouse bronchoalveolar lavage fluid (BALF) cell and lung tissues. Consistent with the progression of fibrosis, there were far more M2 macrophages at the 5th day after ventilation and remained dominant for 2 weeks. High-VT MV induced epithelial-mesenchymal transition (EMT) on day 7, accompanied by the increased expression of TGF-beta 1 and p-Smad2/3. In vitro experiments, the co-culture of M2 macrophage and MLE-12 cells resulted in a significant EMT and upregulation of TGF-beta 1 and p-Smad2/3 in MLE-12 cells. To summarize, our findings suggested the persistent tilt polarization toward M2 macrophages was associated with EMT during the course of ventilator-induced pulmonary fibrosis, which may play its roles through activation of epithelial TGF-beta 1/Smad2/3 signaling.