Background. Premature membrane rupture is a known causes of preterm labor and accounts for approximately one-third of cases. Vitamin D deficiency may play a role in preterm labor as well. Objective. The present study aimed to compare vitamin D levels in mothers and infants with and without prolonged rupture of membranes (PROM). Materials and methods. This cross-sectional study was conducted with 241 babies, with and without a history of membrane rupture, in mothers in the neonatal ward of Ghaem Hospital, Mashhad, from 2019 to 2021 with available sampling methods. After confirmation of prolonged membrane rupture (more than 18 hours before birth) based on history and examination with a speculum, we completed a data collection tool, a checklist including laboratory evaluation and neonatal and maternal characteristics. The conditions of neonates with and without prolonged membrane rupture were compared. Data were analyzed by T-test and Chi-square. Results. We examined a total of 241 neonates including 148 (61.4%) without prolonged rupture of the membranes in mothers and 93 (38.6%) with PROM. There were statistically significant differences between the two groups regarding: maternal vitamin D level (p = 0.001); neonatal vitamin D level (p = 0.001); and fifth minute Apgar score (p = 0.003). These variables were lower in the group of neonates with PROM. Conclusion. Vitamin D deficiency in mothers was significantly associated with prolonged membrane rupture. With increasing severity of vitamin D deficiency, the probability of PROM increases and, therefore, the likelihood of preterm labor and its complications rises.