Hyaluronic acid (HA), a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously demonstrated that both CD44 and TLR4, but predominately TLR4, mediated HA stimulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal mice. Here we address the questions of which cell type expresses the relevant TLR4 in driving intestinal growth and what are the downstream events from TLR4 activation. Studies were done in 14-day-old mice: wild type (WT), mice deficient in cyclooxygenase 2 (COX2), mice deficient in myeloid cell TLR4, and mice deficient in epithelial cell epidermal growth factor receptor (EGFR). Biological end points included crypt fission and Lgr5 cell proliferation. In WT mice, treatment with NS-398 (a COX2 inhibitor), clodronate (a macrophage-depleting agent), or tyrphostin (an EGFR inhibitor) resulted in 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with control mice. Mice deficient in COX2 or myeloid TLR4 or epithelial cell EGFR all had 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with WT mice. Administration of dimethyl PGE2, a stable PGE2 analog, increased crypt fission and Lgr5+ stem cell proliferation. Administration of dimethyl PGE2 reversed the effects of NS-398, clodronate, COX2 deficiency, and myeloid TLR4 deficiency but had no effect on mice treated with tyrphostin or mice deficient in epithelial cell EGFR. We conclude that, in postnatal mice, ~30% of intestinal growth as manifested by crypt fission and Lgr5+ stem cell proliferation is driven by a novel pathway: Extracellular HA binds TLR4 on pericryptal macrophages, inducing the production of PGE2 through COX2. PGE2 transactivates EGFR in Lgr5+ epithelial stem cells, resulting in Lgr5+ stem cell proliferation and crypt fission.NEW & NOTEWORTHY This study, in newborn mice, describes a novel molecular pathway regulating Lgr5+ epithelial stem cell proliferation and normal intestinal elongation, as assessed by crypt fission. In this pathway, endogenous extracellular hyaluronic acid binds to Toll-like receptor 4 on pericryptal macrophages releasing PGE2 which binds to epidermal growth factor receptor on Lgr5+ stem cells resulting in proliferation. Lgr5+ stem cell proliferation leads to crypt fission and intestinal elongation. The demonstration that normal growth requires microbial-independent Toll-like receptor activation is novel.