Background Preterm newborn nutrition affects postnatal skeletal growth and bone mineralization, but studies have not yet fully concluded the relationship between nutrition and osteopenia. This study was intended to investigate the impact of nutritional factors on osteopenia in preterm newborns. Methods This is a case-control study with babies born with gestational age less than or equai to 32 weeks in a high-risk maternity hospital, between 2018 and 2019. The population consisted of 115 newborns, being 46 cases (40%) and 69 controls (60%). Disease outcome was based on serum alkaline phosphatase levels > 900UL/l and hypophosphatemia < 4 mg/dl. Gestational data at birth and clinical and nutritional follow-up data during 8 weeks postnatally were assessed. Variables were assessed using regressive logistic models. Findings Preterm infants who were fed pasteurized fresh human milk with acidity greater than or equal to 4 ºDornic are 5.36 times more likely to develop osteopenia (p = 0.035). Higher calcium intake, compared to controls, also increased the probability of disease occurrence OR 1.05 (CI 1.006-1.1); p = 0.025, while the presence of a partner OR 0.10 (CI 0.02-0.59); p = 0.038 and the shortest time using sedatives OR 0.89 (CI 0.83-0.98); p = 0.010 were protective factors associated with osteopenia. Extremely low birth weight OR 5.49 (CI 1.20-25.1); p = 0.028, sepsis OR 5.71 (CI 1.35-24.2); p = 0.018 and invasive ventilatory support OR 1.09 (CI 1.03-1.18); p = 0.007 were risk factors. Conclusions Acidity and high calcium intake are the main nutritional factors associated with osteopenia of prematurity. Further studies on the use of human milk with lower acidity, recommendation and nutritional supplementation of calcium should be accomplished to guide prevention strategies in newborns at risk for osteopenia during hospital stay. Keywords: Infant, Premature, Bone diseases, Metabolic, Nutritional support