AbstractThe small intestine serves as the first channel of dietary Vitamin A (VA) and the unique organ of VA absorption and metabolism. However, there have not been extensive investigations on the exact mechanisms within VA‐related changes in intestinal metabolic disorders. This research is designed to analyze whether and how VA affects intestinal metabolic phenotypes. Male C57BL/6 mice after weaning were randomly fed a VA control diet (VAC) or a VA‐deficient diet (VAD) during the entire pregnancy and lactation process. After a total of 11 weeks, cohorts of VA deprived were next fed to a VA control diet (VAD‐C) for another 8 weeks. The concentration of retinol was measured by a high‐performance liquid chromatography system. The 16S gene sequencing was used to evaluate the intestinal microbiota changes. Through the use of histological staining, western blots, quantitative PCR, and enzyme‐linked immunosorbent assays, the intestinal morphology, inflammatory factors, and intestinal permeability were all evaluated. Following the decrease of the tissue VA levels, VAD mice show a decrease in tissue VA levels, community differences, and the richness and diversity of intestinal microbiota. VAD diet‐driven changes occur in intestinal microbiota, accompanied by a higher mRNA expression of intestinal inflammatory cytokines and an increase in intestinal permeability. As dietary VA is reintroduced into VAD diet‐fed mice, the tissue VA levels, inflammatory response, and intestinal homeostasis profiles are all restored, which are similar to those found after the occurrence of VA‐controlled changes within intestinal microbiota. VA deficiency caused the imbalance of intestinal metabolic phenotypes through a mechanism involving changes in intestinal microbiota. It is thought that intestinal microbiota metabolic influences represent a new salient and additional mechanism, which can be used as a new method to achieve the onset and treatment of the effect of VAD on intestinal homeostasis impairment.