Background: Current evidence no longer supports the instillation of normal saline (NS) during endotracheal suctioning. However, it is unclear whether and to what extent nurses practice NS instillation in Jordan’s neonatal intensive-care units (NICUs). Purpose: To describe the knowledge and practice of Jordanian nurses regarding NS instillation before endotracheal suctioning. Methods: A cross-sectional descriptive method was used. Data was collected from 136 NICU nurses using a 29-item self-administered questionnaire. The participants were from 7 NICUs of 7 hospitals in Jordan (north, middle and south of Jordan). The questionnaire asked participants about their knowledge and practice regarding endotracheal suctioning. Results: Findings showed a high frequency of NS instillation during endotracheal suctioning. Among the participants, 44.1% believed that NS was beneficial in loosening secretions, 57.4% believed that it could stimulate the cough response, 56.6% believed that it promoted neonate comfort and 41.2% believed that it could reduce bacterial growth. In practice, 80.0% of nurses used open rather than closed suctioning, 57.1% did not use more than 5 ml of NS during suctioning and 92.4%. instilled 0.9% normal saline directly onto the endotracheal tube rather than the in-line suction adapter. Moreover, 53.7% of participants reported that suctioning mechanically ventilated premature neonates out of regularity and not because it was clinically indicated. Conclusion: This is the first study that explored NICU nurses’ knowledge and practice of NS use during endotracheal suctioning in Jordan. Educational programs can be implemented to improve knowledge regarding the complications of NS instillation. Implications for Nursing: NICU guidelines should be implemented to ensure that suctioning practices reflect current standards and education and training programs should be developed to target the knowledge and skills of nurses Keywords: Normal saline instillation, Suctioning, Endotracheal tube, NICUs, Jordan.