For a preferential homing of T cells to the gut, expression of the integrin α4β7 and the chemokine receptor CCR9 is essential and is induced by antigenic stimulation with dendritic cells from the gut-associated lymphoid organs. Here, we show that the vitamin A (retinol) metabolite, retinoic acid, enhances the expression of α4β7 and CCR9 on T cells upon activation and imprints them with the gut tropism. Dendritic cells from the gut-associated lymphoid organs produced retinoic acid from retinol. The enhanced α4β7 expression on T cells by antigenic stimulation with these dendritic cells was suppressed by the retinal dehydrogenase inhibitor citral and the retinoic acid receptor antagonist LE135. Accordingly, vitamin A deficiency caused a reduction in α4β7⁺ memory/activated T cells in lymphoid organs and a depletion of T cells from the intestinal lamina propria. These findings revealed a novel role for retinoic acid in the imprinting of gut-homing specificity on T cells.