Tracheal suction plays an indispensable role in artificial respiration management. However, we speculate that frequent suctioning with excessive negative pressure may result in acute lung injury (ALI). Therefore, in this study, we evaluated the effects of frequent tracheal suction at excessive negative pressure on ALI in rats under artificial ventilation. Tracheal intubation was performed in 4-week-old male Wistar rats. The rats were divided into the following five groups: three suction groups with tracheal suctioning performed for 10 s every 2 min for a total of 30 times at −20 kPa (−20 kPa group), −40 kPa (−40 kPa group), and −90 kPa (−90 kPa group); the artificial ventilation (AV) group with artificial ventilation performed for 1 h following intubation; and the normal (N) group without intubation or artificial ventilation. All rats were euthanized 12 h after the end of tracheal suctioning or artificial ventilation. The lungs were collected and stained with hematoxylin-eosin for histological evaluation. Blood IL-6 and TNF-α levels were measured using ELISA. The mortality rates observed in all five groups up to 3 h after suctioning or artificial ventilation were recorded. In another experiment, tracheal suctioning (−90 kPa) was performed in rats that had received a neutrophil elastase inhibitor (sivelestat), and the lung tissues were evaluated after 24 h. Cytokine levels were also measured. Scores for infiltration of inflammatory cells (mainly white blood cells) in the alveolar space, edema, and hemorrhage in the suction groups was higher than those in the N group. Typical histopathological features of ALI were observed in the −90 kPa group. Blood IL-6 level in the −90 kPa group was higher than those in the other groups. In the rats that received sivelestat, blood IL-6 and TNF-α levels were slightly elevated and the changes in lung tissue were mild after performing tracheal suctioning at −90 kPa. Frequent tracheal suctioning at excessive pressure may induce inflammation in the lung tissue, thereby promoting ALI. In this study, no findings indicating inflammation were observed at −20kPa. Future clinical studies should investigate the appropriate suction pressure and frequency in patients under artificial respiration management.