Background: Early onset sepsis (EOS) is a significant cause of morbidity and mortality among infants. Emerging evidence suggests a potential role of vitamin D in modulating the immune response and reducing infection risk. This study aimed to investigate the association between vitamin D levels and the occurrence of early onset sepsis in infants. Methods: A prospective observational study was conducted on a cohort of 100 infants admitted to the neonatal intensive care unit (NICU) in Silchal Medical College, Silchar between 2017 to 2018, with suspected sepsis. Blood samples were collected within 24 hours of birth to assess serum vitamin D levels using a standardized assay. Infants were followed up for the development of early onset sepsis, and clinical, laboratory, and demographic data were collected. Results: Of the 100 infants included in the study, 25 (25%) were diagnosed with early onset sepsis based on clinical and laboratory criteria. The mean serum vitamin D level in the sepsis group was significantly lower compared to the non-sepsis group (p < 0.05). Infants who developed sepsis had a mean vitamin D level of 15.2 ng/mL (SD ± 4.6), while those without sepsis had a mean level of 22.6 ng/mL (SD ± 6.2). Furthermore, a logistic regression analysis demonstrated that lower vitamin D levels were independently associated with an increased risk of early onset sepsis (OR = 0.78, 95% CI: 0.67–0.92, p = 0.004). After adjusting for potential confounders including gestational age, birth weight, and maternal vitamin D status, the association remained significant (OR = 0.81, 95% CI: 0.69–0.95, p = 0.012). Conclusion: This prospective observational study suggests a significant association between lower serum vitamin D levels and an increased risk of early onset sepsis in infants. Adequate vitamin D supplementation during pregnancy and early infancy may play a crucial role in reducing the susceptibility to early onset sepsis. Further research is needed to elucidate the underlying mechanisms and establish optimal vitamin D supplementation strategies for neonates at risk of sepsis.