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An increasing number of studies have confirmed the role of intestinal microbiota in early life maturation including maturation of intestinal immune function. The interaction of the TLR4 with colonizing bacteria in intestinal development is incompletely understood. Thisstudy investigates the mechanism (s) by which TLR4 interacts with metabolites (postbiotics) of the colonizing bacterium, <i>Bifidobacteria Longum </i>subsp. <i>Infantis (B. Infantis) </i>to determine its effect on intestinal development. By using a fetal human small intestinal cell line, authenticatedby human fetal intestinal organoids, we found that TLR4-mediated postbiotics induced immature enterocyte proliferation and filamentous actin (F-actin) maturation both at the mRNA and protein levels. Oral feeding of wild-type (WT) and TLR4 gene knockout (TLR4 ^-/-) neonatal mice of<i>B.Infantis</i> postbiotics increased proliferation of mRNA levels in wild-type mice but not in TLR4^-/- mice, both in the ileum and colon. Postbioticscan also change tight junction distribution in WT neonatal mouse colon but not in TLR4 ^-/-mice. Our data suggest a novel regulation of intestinal development by a synergistic role of the innate immune receptor TLR4 and early life colonizing bacteria, such as <i>B. Infantis,</i> in both the fetal human and immature mouse intestine. This study should provide new insights into the mechanisms of intestinal maturation as well as opportunities to target novel approaches to NEC prevention and treatment.