MyD88-dependent signaling in myo-/fibroblasts (MFs) is involved in epithelial barrier restoration and tolerance. However, the role of MyD88-dependent signaling by MFs in the regulation of inflammatory responses by macrophages in the colonic mucosa is poorly understood. Because colonic MFs respond to MyD88 activation with production of molecules involved in the regulation of macrophages (PGE2, PD-L1, etc.), we hypothesize that MF mediated MyD88 signaling regulates inflammatory responses from macrophages in the colon. Tamoxifen inducible Col1α2Cre Myd88 floxed mice (fibroblast and myofibroblast-specific MyD88 deletion) and α-SMACre MyD88 floxed mice (myo-fibroblast and smooth muscle cell-specific MyD88 deletion) on the same genetic background (C57BL/6) were used in this study. We observed that deletion of MyD88 within MFs resulted in the inflammatory changes and infiltration of lymphocytes within colonic mucosa and moderately aggravated DSS induced acute colitis. Activation of the lymphocyte trafficking and type 1 inflammatory pathways were observed (RNAseq data) in DSS-treated and non-treated mice lacking MyD88 within MFs. This was associated within increased infiltration of F4/80+ CD11b+ macrophages, but not CD11c+ dendritic cells. CX3CR1high CCR2+cells producing TNF-α were predominant within the macrophage population suggesting increased chemotaxis of inflammatory macrophages to the colonic mucosa. Further, overall increased TNF-α production was observed in the mice lacking MyD88 within MFs and depletion of the macrophages resulted in a decrease in TNF-α levels within the colonic mucosa. Therefore, our data suggest that MF- mediated MyD88 signaling contributes to the maintenance of colonic mucosal homeostasis through suppression of the influx of TNF-α producing inflammatory macrophages.