Background: Endotracheal suctioning is a vital procedure performed by intensive care nurses to maintain airway patency and optimal gas exchange in critically ill patients under mechanical ventilation. As the procedure can cause several complications, intensive care nurses should have adequate evidence-based knowledge on how to perform the procedure. Aims: This study aimed at examining intensive care nurses' knowledge and experience of the closed suctioning system. This study was a prospective, cross-sectional, descriptive, and multi-centered study conducted with 195 nurses working in the tertiary intensive care units in five Turkish hospitals. Subjects and Methods: The data were collected with a questionnaire developed by the researchers in light of the literature. The data were evaluated with Kruskal–Wallis and Mann–Whitney U tests. Results: The mean age of the nurses was 30.58 ± 6.28 years. The mean knowledge score of the nurses for the closed system was 27.35 ± 12.05 (range = 0–80) and 80.5% of them obtained scores ranging between 0 and 40. Also, 71.8% stated that they did not have any difficulty performing suctioning with the closed system catheter, and 85% said that they saved time with the closed system. However, 50.8% of the nurses said they were not able to suction viscous and sticky secretions efficiently, 44.6% stated they did not need to irrigate the inside of the catheter, and 45.1% of them were undecided whether the coude tip catheter led to hemorrhagic secretion more than straight tip catheter. Conclusions: Almost one-fifth of the nurses had good scores on evidence-based knowledge of suctioning. Most of the nurses' experiences regarding the use and manipulation of the closed system were positive. However, almost half of the nurses pointed out that the closed system was not effective enough to suction viscous and sticky secretions. Regular in-service training sessions may help to improve nurses' current knowledge and experiences.