© 2014, Springer-Verlag Berlin Heidelberg.Vitamin D has been linked with immunity, and the immunomodulatory role of this molecule in regulating key elements of the immune system has become an area of intense scientific investigation. We designed a case–control study to investigate whether neonates with early-onset neonatal sepsis (EONS) had lower levels of vitamin D. The primary exposure for the analysis in the study was the cord-blood level of 25-hydroxyvitamin D (25(OH)D). Of the 2571 live births occurring during the 18-month study period, 53 infants were admitted to the neonatal intensive care unit with suspected EONS. After clinical and laboratory confirmation, 40 newborns with EONS and 43 controls were analyzed. Cord-blood 25(OH)D levels of infants in the study group were significantly lower than that of the control group (median 12.6 ng/mL (3.1–78.9) vs. 21 (5–118); p = 0.038, respectively). In multivariate models, a low cord-blood 25(OH)D level (<30 ng/ml) was associated with an increased risk of EONS (OR = 5.6; 95 % CI = 1.3–23.5). Conclusion: Cord-blood 25(OH)D levels of neonates with EONS were significantly lower than that of the healthy controls, and a low level of cord-blood vitamin D was found to be associated with an increased risk of EONS. Further studies are warranted to confirm this association.