Breast milk prebiotic oligosaccharides are believed to promote enteral tolerance. Many mothers delivering preterm are unable to provide sufficient milk. We conducted a multicenter, randomized, controlled trial comparing preterm formula containing 0.8 g/100 mL short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides in a 9:1 ratio and an otherwise identical formula, using formula only to augment insufficient maternal milk volume. Infants were randomized within 24 h of birth. The primary outcome (PO) was time to establish a total milk intake of 150 mL/kg/d PO and the principal secondary outcome (PSO) was proportion of time between birth and 28 d/discharge that a total milk intake of ≥ 150 mL/kg/d was tolerated. Other secondary outcomes included growth, fecal characteristics, gastrointestinal signs, necrotizing enterocolitis, and bloodstream infection. Outcomes were compared adjusted for prespecified covariates. We recruited 160 infants appropriately grown for GA <33 wk. There were no significant differences in PO or PSOs. After covariate adjustment, we showed significant benefit from trial formula in PSO with increasing infant immaturity (2.9% improved tolerance for a baby born at 28-wk gestation and 9.9% at 26-wk gestation; p < 0.001) but decreased or no benefit in babies >31-wk gestation. Prebiotic supplementation appears safe and may benefit enteral tolerance in the most immature infants.