Serotype 4b strains of Listeria monocytogenes have been responsible for most large outbreaks of listeriosis. In L. monocytogenes serotype 4b, gtcA and gltA have been implicated in serotype-specific glycosylation of the teichoic acid of the cell wall with galactose and glucose. In this study, we investigated the impact of mutations in gltA (resulting in absence of glucose on teichoic acid) and gtcA (resulting in absence of galactose, and markedly reduced glucose on teichoic acid) on virulence following intragastric infection of anesthetized A/J mice. The gltA mutant was not impaired in virulence in this model. In contrast, testing of gtcA mutants constructed in two different strains showed that the mutants were recovered in lower numbers than their respective parent strains from the spleen, liver, ceca, and gall bladders of intragastrically inoculated mice. Genetic complementation of the gtcA mutation partially restored gastrointestinal virulence. When mice were inoculated intravenously, the gtcA mutants were also recovered in lower numbers from the liver (for both mutant strains) and the spleen (for one mutant strain) than their respective parental strains. The mutants were also evaluated for invasion and intracellular multiplication in the Caco-2 human intestinal epithelial cell line. Inactivation of gltA did not affect invasion or intracellular growth of the bacteria. In contrast, gtcA mutants showed decreased invasion, but normal multiplication in Caco-2 cells. Overall, these data demonstrate a role for gtcA in the pathogenesis of gastrointestinal listeriosis in mice, and suggest that diminished ability of gtcA mutants to invade intestinal epithelial cells may be partly responsible for decreased gastrointestinal virulence.