Gut commensal microbes affect the development and activation of the mucosal and systemic immune systems. However, the exact molecular mechanism of these microbes that is involved in the development of colitis remains unclear. The present study was conducted to determine the distinct role of the innate immune system in the development of a dextran sulfate sodium (DSS) colitis model in MyD88−/− mice, because myeloid differentiation protein (MyD88) is a major adaptor molecule essential for signaling via Toll-like receptors (TLRs). To this end, MyD88−/− and wild-type (WT) mice received sterile distilled water containing 1.2% DSS for 8 days. The survival rate, total clinical score (body weight loss, stool consistency, and rectal bleeding), colon length, and histological score were assessed. The expression of surface markers (F4/80 and CD4) on infiltrating lamina propria mononuclear cells was analyzed immunohistochemistrically. MyD88−/− mice exhibited increased susceptibility to DSS-induced colitis, as reflected by significantly higher lethality and higher clinical and histological scores, and more severe colonic shortening compared to WT mice. Immunohistochemical analysis revealed a significant increase of both F4/80+ macrophages and CD4+ T cells in the inflamed mucosa in DSS-fed MyD88−/− mice compared to DSS-fed WT mice. These findings suggest that, via MyD88 signaling, the innate immune system in the gut plays an important protective role in colitis.