Recent studies suggest that vitamin D deficiency is associated with intestinal dysfunctions, but the underlying mechanism remains unclear. This study aimed to investigate whether maternal vitamin D deficiency increases intestinal permeability in offspring and its related mechanism. Timed-pregnant mice were fed with either a standard chow diet (SC) or a vitamin D-deprived chow diet (VD-) 6 weeks prior to breeding and kept on the same diet until the end of gestation. All offspring were fed an SC for 3 weeks after weaning and then observed for effects associated with maternal vitamin D deficiency. Maternal vitamin D deficiency increased intestinal permeability in offspring, which corresponded with the decreased expression of the tight junction protein claudin-1. Maternal vitamin D deficiency also repressed the messenger RNA expression of wingless/integrated family member 3a (Wnt3a) and the protein expression of nuclear β-catenin. The decreased Wnt3a gene expression in male was concurrent with the changes in histone H4 acetylation at either promoter or coding regions. The activation of the Wnt/β-catenin pathway protected against the impairment of intestinal permeability induced by maternal vitamin D deficiency. Maternal vitamin D deficiency increased intestinal permeability and decreased tight junction protein expression in offspring. The suppression of the Wnt/β-catenin signaling pathway through histone modification might be involved in the underlying mechanism.