Lymphocyte inhibitory factor A (lifA) in Citrobacter rodentium encodes the large toxin lymphostatin, which contains two enzymatic motifs associated with bacterial pathogenesis, a glucosyltransferase and a protease. Our aim was to determine the effects of each lymphostatin motif on intestinal epithelial-barrier function. In-frame mutations of C. rodentium lifA glucosyltransferase (CrGlM21) and protease (CrPrM5) were generated by homologous recombination. Infection of both model intestinal epithelial monolayers and mice with C. rodentium wild type resulted in compromised epithelial barrier function and mislocalization of key intercellular junction proteins in the tight junction and adherens junction. In contrast, CrGlM21 was impaired in its ability to reduce barrier function and influenced the tight junction proteins ZO-1 and occludin. CrPrM5 demonstrated decreased effects on the adherens junction proteins beta-catenin and E-cadherin. Analysis of the mechanisms revealed that C. rodentium wild type differentially influenced Rho GTPase activation, suppressed Cdc42 activation, and induced Rho GTPase activation. CrGlM21 lost its suppressive effects on Cdc42 activation, whereas CrPrM5 was unable to activate Rho signaling. Rescue experiments using constitutively active Cdc42 or C3 exotoxin to inhibit Rho GTPase supported a role of Rho GTPases in the epithelial barrier compromise induced by C. rodentium. Taken together, our results suggest that lymphostatin is a bacterial virulence factor that contributes to the disruption of intestinal epithelial-barrier function via the modulation of Rho GTPase activities.