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The aim of the study was to assess the effect of medically graded enteral honey supplementation on the intestinal microbiota, immune response, and somatic growth of preterm infants.A prospective randomized controlled trial was conducted on preterm infants with gestational age ≤34 weeks and postnatal age >3 days. After reaching 1/2 goal enteral feeds, medically graded bee honey was added to milk at a dose of 5, 10, 15, and 0 g/day for 2 weeks in groups A, B, C, and D, respectively. Anthropometric measurements, CD4 and CD8 cytokines, stool cultures, and stool polymerase chain reaction assays for molecular detection of microbiomes were performed at 0, 7, and 14 days of intervention. Analysis of variance test was used to detect differences among the 4 groups.A total of 40 subjects were enrolled; 10 in each arm of the study. Compared with group D, all 3 intervention groups demonstrated significant increase in weight (P < 0.0001). Head circumference increased in groups B and C (P = 0.0056). There were no changes in CD4 or CD8 cytokines (P = 0.24 and P = 0.11, respectively). Enterobacter stool colonization decreased in groups A and B (P = 0.002), whereas Bifidobacterium bifidum colony counts increased in groups A, B, and C (P = 0.002) and lactobacilli colony counts increased in group B (P < 0.0001). Applying real-time polymerase chain reaction, B bifidum and lactobacilli increased in group C (P < 0.0001).Supplementation of milk formula with medically graded honey was associated with changes in physical growth and colonic microbiota of preterm infants. Further studies are needed to examine the sustainability of these effects and associated long-term outcomes.