Purpose: Premature infants receiving probiotics have a decreased incidence of necrotizing enterocolitis. This may be mediated by intestinal bacterial signaling via toll-like receptors (TLRs) 2 and 4 maintaining intestinal homeostasis. We hypothesized that TLRs 2 and 4 are protective against ischemia-reperfusion (I/R) intestinal injury. Methods: Two-week-old C57BL/6 wild-type (WT), B6.TLR2-/-, B6.TLR4-/-, B6.TLR2-/-4-/-, and microbially reduced (antibiotic-treated) mice (MR) underwent 60 minutes of superior mesenteric artery occlusion (I) followed by 90 minutes of reperfusion (R). Small intestine was harvested for analysis of microscopic injury, apoptosis, and inflammatory gene expression using quantitative polymerase chain reaction. Results: After I/R, the median histologic injury scores of the B6.TLR4-/-, B6.TLR2-/-4-/-, and MR pups were higher than the WT or B6.TLR2-/- pups that corresponded with greater apoptosis based on terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling and activated caspase-3 immunostaining. B6.TLR4-/-, B6.TLR2-/-4-/-, and MR also had elevated tissue innate immunity-associated chemokine and cytokine expression. Conclusions: Neonatal mice deficient in TLR4, either alone or also deficient in TLR2, as well as those lacking a normal commensal intestinal microbiome are more susceptible to an I/R model of intestinal injury. These results may provide a mechanism for commensal bacterial-mediated protection, which may help to direct further studies to elucidate the mechanism of probiotic protection. © 2010 Elsevier Inc. All rights reserved.