To investigate the efficiency of closed tracheal suction system (CTSS) using novel splash-proof ventilator circuit component on ventilator-associated pneumonia (VAP) and the colonization of multiple-drug resistant bacteria (MDR) in patients undergoing mechanical ventilation (MV) prevention. A prospective single-blinded randomized parallel controlled intervention study was conducted. 330 severe patients admitted to the intensive care unit (ICU) of the First Hospital of Jiaxing from January 2014 to May 2016 were enrolled, and they were divided into open tracheal suction group, closed tracheal suction group, and splash-proof suction group on average by random number table. The patients in the three groups used conventional ventilator circuit component, conventional CTSS, and CTSS with a novel splash-proof ventilator circuit component for MV and sputum suction, respectively. The incidence of VAP, airway bacterial colonization rate, MDR and fungi colonization rate, duration of MV, length of ICU and hospitalization stay, and financial expenditure during hospitalization, as well as the in-hospital prognosis were recorded. After excluding patients who did not meet the inclusion criteria, incomplete data, backed out and so on, 318 patients were enrolled in the analysis finally. Compared with the open tracheal suction group, the total incidence of VAP was decreased in the closed tracheal suction group and splash-proof suction group [20.95% (22/105), 21.90% (23/105) vs. 29.63% (32/108)], but no statistical difference was found (both P > 0.05), and the incidence of VAP infections/1 000 MV days showed the same change tendency (cases: 14.56, 17.35 vs. 23.07). The rate of airway bacterial colonization and the rate of MDR colonization in the open tracheal suction group and splash-proof suction group were remarkably lower than those of closed tracheal suction group [32.41% (35/108), 28.57% (30/105) vs. 46.67% (49/105), 20.37% (22/108), 15.24% (16/105) vs. 39.05% (41/105)] with significantly statistical differences (all P < 0.05). Besides, no significantly statistical difference was found in the fungi colonization rate among open tracheal group, closed tracheal group, and splash-proof suction group (4.63%, 3.81% and 6.67%, respectively, P > 0.05). Compared with the closed tracheal suction group, the duration of MV, the length of ICU and hospitalization stay were shortened in the open tracheal suction group and splash-proof suction group [duration of MV (days): 8.00 (4.00, 13.75), 8.00 (5.00, 13.00) vs. 9.00 (5.00, 16.00); the length of ICU stay (days): 10.00 (6.00, 16.00), 11.00 (7.00, 19.00) vs. 13.00 (7.50, 22.00); the length of hospitalization stay (days): 16.50 (9.25, 32.00), 19.00 (10.50, 32.50) vs. 21.00 (10.00, 36.00)], and financial expenditure during hospitalization was lowered [10 thousand Yuan: 4.95 (3.13, 8.62), 5.47 (3.84, 9.41) vs. 6.52 (3.99, 11.02)] without significantly statistical differences (all P > 0.05). Moreover, no significantly statistical difference was found in the in-hospital prognosis among the three groups. CTSS performed using novel splash-proof ventilator circuit component shared similar advantages in preventing VAP with the conventional CTSS. Meanwhile, it is superior because it prevented the colonization of MDR and high price in the conventional CTSS.Clinical Trail Registration Chinese Clinical Trial Registry, ChiCTR-IOR-16009694.