Very low birth weight (VLBW) preterm newborns lack some nutrients such as vitamin D (VD), which is important in the function and development of different systems. Objective: To evaluate serum levels of 25-OH-VD in VLBW newborns and to describe the possible association between its deficit and frequent morbidities in this population. Patients and Methods: Cross-sectional study of VLBW newborns (< 1,500 g and/or < 32 weeks of gestational age). A single measurement of serum 25-OH-VD levels was performed in the first 72 hours of postnatal life (chemiluminescent immunoassay). Perinatal characteristics, treatments performed, and frequent comorbidities were analyzed. Deficiency of 25-OH-VD was defined as levels & LE; 20 ng/ml, determining a statistical association between this and various comorbidities at hospital discharge in the neonatal period. Results: 46 preterm newborns were evaluated. The mean serum level of 25-OH-VD was 19.7 & PLUSMN; 6.7 ng/ml. 52.2% (24/46) presented deficient levels, with lower values observed at lower gestational age (p = 0.01). There was an association between 25-OH-VD deficiency, the need for conventional mechanical ventilation (p = 0.04), and longer hospital stay (p < 0.01). There was an association with the presence of hemodynamically significant ductus arteriosus (p < 0.01). Conclusions: Deficiency of 25-OH-VD was frequent in VLBW newborns, with lower levels at lower gestational age. There was an association between 25-OH-VD deficiency, hospital stay, need for respiratory support, and patent ductus arteriosus.