Retinoic acid (RA) produced by intestinal dendritic cells (DCs) imprints gut-homing specificity on lymphocytes and enhances Foxp3⁺ regulatory T-cell differentiation. The expression of aldehyde dehydrogenase (ALDH) 1A in these DCs is essential for the RA production. However, it remains unclear how the steady-state ALDH1A expression is induced under specific pathogen-free (SPF) conditions. Here, we found that bone marrow-derived dendritic cells (BM-DCs) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) expressed Aldh1a2, an isoform of Aldh1a, but that fms-related tyrosine kinase 3 ligand-generated BM-DCs did not. DCs from mesenteric lymph nodes (MLN) and Peyer's patches (PP) of normal SPF mice expressed ALDH1A2, but not the other known RA-producing enzymes. Employing a flow cytometric method, we detected ALDH activities in 10–30% of PP-DCs and MLN-DCs. They were CD11c^highCD4^−/lowCD8α^intermediateCD11b^−/low F4/80^{low/intermediate}CD45RB^lowCD86^highMHC class II^highB220⁻CD103⁺. Equivalent levels of aldehyde dehydrogenase activity (ALDHact) and ALDH1A2 expression were induced synergistically by GM-CSF and IL-4 in splenic DCs in vitro. In BM-DCs, however, additional signals via Toll-like receptors or RA receptors were required for inducing the equivalent levels. The generated ALDH1A2⁺ DCs triggered T cells to express gut-homing receptors or Foxp3. GM-CSF receptor-deficient or vitamin A-deficient mice exhibited marked reductions in the ALDHact in intestinal DCs and the T cell number in the intestinal lamina propria, whereas IL-4 receptor-mediated signals were dispensable. GM-CSF⁺CD11c⁻F4/80⁺ cells existed constitutively in the intestinal tissues. The results suggest that GM-CSF and RA itself are pivotal among multiple microenvironment factors that enable intestinal DCs to produce RA.