Objective To explore the application effect of ICU checklist of medical cooperation on prevention of ventilator-associated pneumonia. Methods Totally 728 patients with mechanical ventilation from July 2015 to July 2016 in ICU were assigned to the experimental group with using of the checklist; and 685 patients with mechanical ventilation from June 2014 to June 2015 were set as the control group who did not use the checklist. The incidence of ventilator-associated pneumonia and medical cooperation were compared before and after the implementation of the checklist. Results The incidence of ventilator associated pneumonia were 6.72% (46/685) and 3.02% (22/728), the intervention strategy execution rates were 63.36% (434/685) and 84.89% (618/728) before and after the application of checklist, there was significant difference between two groups (χ2=10.51, 86.03, P<0.01). Medical cooperation (doctors and nurses) and satisfaction scored 18.53 ± 2.39, 21.91 ± 2.16, 30.93 ± 2.18 and 26.71 ± 2.21, 28.64 ± 2.55, 45.33 ± 1.74 before and after the application of checklist, there was significant difference between two groups (t=6.14, 14.25, 2.39, P<0.01 or 0.05). Conclusions The application of checklist can improve the medical cooperation on prevention of ventilator-associated pneumonia management, reduce the incidence of ventilator-associated pneumonia, improve medical cooperation, nursing satisfaction and the management efficiency, which is worthy of popularization and application.