Retinoic acid (RA), a well-known vitamin A metabolite, mediates inhibition of the IL-6-driven induction of proinflammatory Th17 cells and promotes anti-inflammatory regulatory T cell generation in the presence of TGF-beta, which is mainly regulated by dendritic cells. To directly address the role of RA in Th17/regulatory T cell generation in vivo, we generated vitamin A-deficient (VAD) mice by continuous feeding of a VAD diet beginning in gestation. We found that a VAD diet resulted in significant inhibition of Th17 cell differentiation in the small intestine lamina propria by as early as age 5 wk. Furthermore, this diet resulted in low mRNA expression levels of IL-17, IFN regulatory factor 4, IL-21, IL-22, and IL-23 without alteration of other genes, such as RORgammat, TGF-beta, IL-6, IL-25, and IL-27 in the small intestine ileum. In vitro results of enhanced Th17 induction by VAD dendritic cells did not mirror in vivo results, suggesting the existence of other regulation factors. Interestingly, the VAD diet elicited high levels of mucin MUC2 by goblet cell hyperplasia and subsequently reduced gut microbiome, including segmented filamentous bacteria. Much like wild-type mice, the VAD diet-fed MyD88-/-TRIF-/- mice had significantly fewer IL-17-secreting CD4+ T cells than the control diet-fed MyD88-/-TRIF-/- mice. The results strongly suggest that RA deficiency altered gut microbiome, which in turn inhibited Th17 differentiation in the small intestine lamina propria.