Objectives: Prematurity and related respiratory distress syndrome (RDS) is the major cause of mortality among neonates in India. Although some studies were showing an association between vitamin D and lung maturation, conclusive evidence was not found yet. The objective of this study was to assess the serum vitamin D in preterm neonates and to determine its possible association with RDS. Material and Methods: A case–control study in which the preterm neonates <37 weeks of age and <2 kg birth weight admitted to neonatal intensive care unit and in postnatal wards in a tertiary care center were included and divided as cases-those with RDS and controls-those without RDS. Two milliliters of whole blood samples were collected from the study population for 25 (OH) D levels. Results: (1) RDS was predominantly seen in <32 weeks (<30 and 30–31 weeks) when compared with the other two gestational age groups. (2) The mean serum vitamin D levels in cases (RDS) were lower compared to controls (non-RDS) (6.97 ± 3.24 in cases vs. 17.08 ± 4.15 in controls). (3) A significant difference in vitamin D levels was obtained among those on a mechanical ventilator and received surfactant administration compared to those who were not on a mechanical ventilator or administered surfactant (P = 0.001 and 0.003). (4) A significant association between vitamin D and adequacy of maternal vitamin D-rich food intake and sunlight exposure was found to be present (P < 0.001 and 0.018). (5). Serum levels of vitamin D were found to be lower (3.3 ± 1.6 ng/mL) in cases who died during the hospital stay compared to those who survived (8.4 ± 2.6 ng/mL), (P < 0.001). Conclusion: A significant difference in vitamin D concentration between preterms with respiratory distress syndrome and those without RDS was reflected in our study. And we also noted low serum vitamin D levels associated with poor outcome.