Hyaluronic acid, a glycosaminoglycan in the extracellular matrix, binds to CD44 and TLR4. We previously addressed the role of hyaluronic acid in small intestinal and colonic growth in mice. We addressed the role of exogenous hyaluronic acid by giving hyaluronic acid IP and the role of endogenous hyaluronic acid by giving PEP-1, a peptide that blocks hyaluronic acid binding to its receptors. Exogenous hyaluronic acid increased epithelial proliferation but had no effect on intestinal length. PEP-1 resulted in a shortened small intestine and colon and diminished epithelial proliferation. In the current study we sought to determine if the effects of hyaluronic acid on growth were mediated by signaling through CD44 or TLR4 by giving exogenous hyaluronic acid or PEP-1 twice a week from 3-8 weeks of age to wild type, CD44^-/- and TLR4^-/- mice. These studies demonstrated that signaling through both CD44 and TLR4 were important in mediating the effects of hyaluronic acid on growth in the small intestine and colon. Extending our studies to early postnatal life we assessed the effects of exogenous hyaluronic acid and PEP-1 on Lgr5⁺ stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5⁺ reporter mice from postnatal day 7 to day 14 decreased Lgr5⁺ cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5⁺ stem cell proliferation, crypt fission and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4.