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Purpose: This study aimed to develop a direct breastfeeding protocol for premature infants admitted to neonatal intensive care units (NICUs) and investigate its efficacy., Background: Direct breastfeeding increases the amount and duration of breastfeeding. However, NICUs have low direct feeding rates owing to medical staff anxiety, lack of knowledge and experience, and fear of overwork. Accordingly, this study developed a protocol for direct breastfeeding in the NICU and evaluated its effect., Methods: The protocol was developed through a literature review, expert validation, and preliminary investigation. Its application effects were identified using a nonexperimental, evidence-based research design targeting premature infants, their mothers, and NICU nurses., Results: The protocol comprised 5 areas and 23 items. Application of the protocol resulted in continuous weight gain of the infants and increased self-efficacy in the mothers' direct breastfeeding ( t = 3.219, P = .004). Significant increases were noted in NICU nurses' direct breastfeeding activities ( t = 3.93, P < .001), breastfeeding rates in the NICU ( P = .037), and direct breastfeeding rates ( P = .007)., Conclusions: Results underscore the value of an evidence-based protocol for improving breastfeeding rates in premature infants. This study highlights the need for continuous nursing education on protocol applications and human resource support., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)