Background: Neonatal ventilator-associated pneumonia (VAP), which involves invasive mechanical ventilation, is a neonatal nosocomial lower airway infection that manifests after 48 hours. The current study aimed to determine the relationship between the development of VAP in intubated newborns and the serum 25- hydroxyvitamin D3 level. Methods: This was a prospective cohort study that included 48 neonates admitted to the Neonatal Intensive Care Unit (NICU) at the Pediatrics Department, Zagazig University Hospital. The patients were divided into 2 groups, the VAP group and the Non-VAP group, with 24 patients in each group. All neonates were subjected to complete history taking including personal data: age and gender, complaint, present, past and perinatal history, developmental and dietetic history, vaccination history and family history and full clinical examination either general examination or local examination for all systems by inspection, palpation, percussion, and auscultation. Results: In our study, in comparison to the Non-VAP group, the VAP group had considerably less 25(OH) vitamin D3 (29.8 ng/ml). Our research revealed a strong positive correlation between vitamin D3 and weight, Apgar score 1 m and 5 min, and PaO2, while there was a significant negative correlation between duration of NICU stay and mechanical ventilation (MV), White blood cells (WBCs), C-reactive protein (CRP) and PaCO2. In our study, at of 29.8, vitamin D3 showed 92.3% sensitivity and 100% specificity, with a p value <0.001. Conclusion: Insufficient serum levels of vitamin D3 may increase the likelihood of developing ventilator-associated pneumonia in neonates undergoing mechanical ventilation.