Proper colonic injury response requires myeloid-derived cells and Toll-like receptor/Myd88 signaling. However, the precise role of Myd88 signaling specifically in myeloid-derived cells that occurs during tissue damage is unclear. Therefore, we created a mouse line with Myd88 expression restricted to myeloid lineages (Myd88(-/-); LysM(Cre/+); ROSA26(Myd88/+); herein Mlcr). In these mice, Myd88 was appropriately expressed and mediated responses to bacterial ligand exposure in targeted cells. Importantly, the severe colonic epithelial phenotype observed in dextran sodium sulfate-injured Myd88(-/-) mice was rescued by the genetic modification of Mlcr mice. During injury, myeloid cell activation and enrichment of Ptsg2-expressing stromal cells occurred within the mesenchyme that surrounded the crypt bases of Mlcr and Myd88(+/-) mice but not Myd88(-/-) mice. Interestingly, these cellular changes to the crypt base mesenchyme also occurred, but to a lesser extent in uninjured Mlcr mice. These results show that Myd88 expression in myeloid cells was sufficient to rescue intestinal injury responses, and surprisingly, these cells appear to require an additional Myd88-dependent signal from a non-myeloid cell type during homeostasis.