The development of antibiotic resistance is associated with high morbidity and mortality, particularly in the intensive care unit (ICU) setting. We evaluated the effect of an antibiotic rotation programme on the incidence of ventilator-associated pneumonia (VAP) caused by antibiotic-resistant Gram-negative bacteria. We conducted a 2-year before-and-after study at two medical-surgical ICUs at two different tertiary referral hospitals. We included all mechanically ventilated patients admitted for ≥48 h who developed VAP. . Conversely, the number of VAP caused by Pseudomonas aeruginosa passed from 8.35 per 1,000 days of MV in P1 to 2.33 per 1,000 days of MV in P2 (p=0.02). No difference in ICU mortality and crude in-hospital mortality between P1 and P2 was noted. Moreover, no significant change of microbial flora isolated through clinical cultures was observed. We were able to conclude that, despite global microbial flora not being affected by such a programme, antibiotic therapy rotation may reduce the incidence of VAP caused by antibiotic-resistant Gram-negative bacteria in the ICU, such as Pseudomonas aeruginosa. The application of this programme may also improve antibiotic susceptibility. However, further studies are needed to confirm our results.