Intestinal microfold (M) cells are critical for sampling antigen in the gut and initiating the intestinal mucosal immune response. In this study, we found that the differentiation efficiency of M cells was closely related to the colitis severity. The expression levels of M cells differentiation-related genes were synchronized with the kinetics of pro-inflammatory cytokines expression originated from dextran sulfate sodium (DSS) induction and Salmonella infection. Compared with wild-type (WT) mice, MyD88 -/- mice exhibited significantly lower expression levels of M cells differentiation-related genes. However, DSS could induce colitis in MyD88 -/- mice but failed to promote M cells differentiation. Furthermore, the receptor activator of the Nuclear Factor-κB ligand (RANKL) induced M cells differentiation in murine intestinal organoids prepared from both WT and MyD88 -/- mice. However, less M cells differentiation were found in MyD88 -/- mice as compared with WT mice. Hence, we concluded that myeloid differentiation factor 88 (MyD88) is an essential molecule for colitis- and RANKL-related M cells differentiation.