The mucosae represent the first line of defense against the invasion of most pathogens, and the mucosal immune system plays a crucial role in the control of infection. Mucosal vaccination can trigger both humoral and cell-mediated immune responses mucosally as well as systemically. Hence, protective immune responses can be elicited effectively by mucosal vaccination. Microfold (M) cells being unique to the mucosal immune system can take up luminal antigens and initiating antigen-specific immune responses. The number of antigen uptake by M cells is directly related to the immune efficacy of mucosal vaccines. Utilizing M cell ligands, M cells-targeting antigen delivery can achieve highly effective mucosal immune responses. The strategy of targeted delivery of antigens to M cells and its applications can be used for the improvement of mucosal immune responses and the development of mucosal vaccines. Despite these efforts, successful development of safe and effective mucosal vaccines remains a big challenge and needs a long way to go, and provably still resort to further researches on cellular properties and functions as well as mucosal immune mechanisms.