Abstract More than 75% of premature infants are given antibiotics in the first week of life. This is lifesaving for neonates with serious bacterial infections but is associated with increased risks of necrotizing enterocolitis (NEC). Given the association of intestinal macrophages (Mϕ) in the pathogenesis in NEC, we characterized the effect of antibiotics on this cell type. Pups were exposed to ampicillin and neomycin via the dam from postnatal day 1 (P1) to P7/8. Mϕ were identified as CD45+ CX3CR1+ CD11b+ F480+ using flow cytometry. In the small intestine (SI) there was an increase in the percentage of Mϕ in antibiotic-exposed lamina propria, but no difference in the proportion of F480-myeloid cells to Mϕ. The percentage of Mϕ in exposed colonic lamina propria cells was unchanged, but the proportion of Mϕ to F480-myeloid cells was increased. This suggests that antibiotic exposure expands myeloid cells in both the SI and colon, but likely via different mechanisms. To further characterize the phenotype of the intestinal Mϕ, we assessed polarity by immunoassay of 13 Mϕ associated cytokines. There were no significant differences in the proximal small intestine cytokine expression between control and antibiotic-exposed tissue. In the distal SI, antibiotic exposed mice had higher levels of TGFβ and CCL2, M2 Mϕ markers. In the colon, control tissue had elevated M1 Mϕ markers compared to antibiotic-exposed mice. Antibiotic-exposed colon had significantly increased levels of TGFβ. These data demonstrate that antibiotic exposure alters Mϕ proportion and polarization. Further work will be necessary to determine how this initial shift away from pro-inflammatory polarization leaves antibiotic-exposed infants at risk for intestinal inflammatory diseases. Supported by NIH T32DK077653