Background/Objectives: The aim of this study was to research and draw conclusions about the effect of a parenteral nutrition (PN) fat emulsion, rich in o-3 fatty acids, on the antioxidant markers of preterm infants, when compared with a standard fat emulsion. This was a double-blind, parallel-group study conducted in Athens, Greece, using an equal randomization method. Subjects/Methods: Thirty-eight infants were selected using a double-blind method and a computer-generated randomization list. Both groups received PN, based on the same protocols. Group A received SMOFlipid fat emulsion, while group B received the standard fat emulsion (Intralipid). Serum levels of vitamin A, E and total antioxidant potential (TAP) were measured on days 0, 7 and 14 of PN support. Clinical and biochemical data were collected on days 0, 14 and on the day of discharge. Results: Serum levels of vitamin E and A were significantly increased in group A, while only vitamin A serum level was increased in group B on the fourteenth day (group A: vitamin E: P-value ¼ 0.002, vitamin A: P-value ¼ 0.000, group B: vitamin E: P-value ¼ 0.065, vitamin A: P-value ¼ 0.000). TAP was increased only in the intervention group (group A: P-value ¼ 0.000, group B: P-value ¼ 0.287). Mild anemia was developed in both groups, while no differences were detected in the infection rate, days of hospitalization, days of ventilator support and days of phototherapy. Conclusions: Oxidative stress was significantly reduced in those neonates fed with o-3 fatty acids, whereas no effect was observed in the neonates fed with standard lipids. Intervention had no effect on infants' growth and clinical outcome.