Background: Considering their special circumstances, premature infants must be taken to a special care and stay long in hospital. Evidence-based care guidelines as the gold standard have an effective role to promote healing and decrease the infants' length of stay. The aim of this study was to determine the effect of education and implementation of evidence-based nursing guidelines on preterm infants' length of stay in NICU. Materials and Methods: This clinical trial was done on a total number of 100 preterm infants, divided into two 50-one groups of intervention and control, hospitalized in Baqiyatallah Hospital of Tehran, in 2014. Data collection was done by means of the Infant Demographic Questionnaire and a researcher-made checklist to record infants' length of hospital stay. Before intervention and two months after implementation of the guidelines, infants’ length of stay was recorded and compared. Data were analyzed by SPSS 16 software and use of descriptive statistics (mean and standard deviation) and inferential tests (t-test, Chi-Square test). Results: The two groups showed no significant difference in age and sex. Average infant's length of stay was 17.9 ± 4.5 days in the control group and 16.8 ± 4.7 days in the intervention group. Finally, infants’ length of stay in the intervention group was less than the control group after a period of two months, which was statistically significant (P = 0.001). Conclusion: The results of this study showed that implementation of evidence-based guidelines is an effective and economical method in infants’ length of stay. Therefore, it is recommended to the authorities and managers of the hospitals and educational centers of the healthcare services to put education and implementation of designed evidence-based guidelines in the priority of their work plans.