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 V chains, and to date two populations of such canonical NKT cells are known in mice, those that express V14 and those that express V7.2. Both populations are selected by non-polymorphic MHC class I-like antigen presenting molecules expressed by hematopoietic cells in the thymus: CD1d for V14 expressing NKT cells and MR1 for those expressing V7.2. The more intensely studied V14 NKT cells have been implicated in diverse immune reactions, including immune regulation and inflammation in the intestine, and the V7.2 expressing cells are most frequent in the lamina propria. Populations of canonical NKT cells can be found in humans that are highly similar in terms of the expression of homologous, invariant TCR 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.