Lymphocytes that combine features of T cells and natural killer (NK) cells are named natural killer T (NKT) cells. The majority of NKT cells in mice bear highly conserved invariant Vα chains, and to date two populations of such canonical NKT cells are known in mice: those that express Vα14 and those that express Vα7.2. Both populations are selected by nonpolymorphic major histocompatibility complex class I-like antigen-presenting molecules expressed by hematopoietic cells in the thymus: CD1d for Vα14-expressing NKT cells and MR1 for those cells expressing Vα7.2. The more intensely studied Vα14 NKT cells have been implicated in diverse immune reactions, including immune regulation and inflammation in the intestine; the Vα7.2 expressing cells are most frequently found in the lamina propria. In humans, populations of canonical NKT cells are found to be highly similar in terms of the expression of homologous, invariant T cell antigen-receptor α-chains, specificity, and function, although their frequency differs from those in the mouse. In this review, we will focus on the role of both of these canonical NKT cell populations in the mucosal tissues of the intestine.