Vitamin A and its derivatives have been shown to regulate the growth and differentiation of gastrointestinal epithelial cells; in addition, vitamin A deficiency has been convincingly shown to be associated with increased susceptibility to infection. The gastrointestinal mucosal barrier, which is a component of the innate immune system, is considered the first line of defense, as it provides a barrier between the external environment and the internal milieu. A disturbance in the integrity of the intestinal epithelium is one of the main factors involved in increased incidence of infections during vitamin A deficiency. In this study, the effects of vitamin A deficiency on microbial ecology and the expression of genes related to the intestinal mucosa's innate immunity were examined in a rat model. Using the 16s rDNA method, we demonstrate that a vitamin A-deficient (VAD) diet increases the total amount of bacteria in the gastrointestinal tract and alters the intestinal microflora. Results show a decrease in the relative proportion of Lactobacillus spp. and the simultaneous appearance of Escherichia coli strains. Lack of vitamin A significantly changed mucin (MUC) dynamics, as reflected by the enlarged goblet-cell "cup" area relative to controls; decreased MUC2 mRNA expression in the jejunum, ileum and colon of VAD rats and increased MUC3 mRNA expression in the ileum and colon of these rats. In addition, vitamin A deficiency down-regulated defensin 6 mRNA expression while up-regulating toll-like receptors 2 and 5 mRNA expressions. The current study indicates that vitamin A deficiency interferes with the integrity of the gastrointestinal mucosal barrier.