EspG is a conserved protein encoded by the locus of enterocyte effacement (LEE) of attaching and effacing (A/E) pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli and Citrobacter rodentium. EspG is delivered into infected host cells by a type III secretion system. The role of EspG in virulence has not yet been defined. Here we describe experiments that probe the virulence characteristics and biological activities of EspG in vitro and in vivo. A C. rodentium espG mutant displayed a significantly reduced ability to colonize C57BL/6 mice and to cause colonic hyperplasia. Epitope-tagged EspG was detected in the apical regions of infected colonic epithelial cells in infected mice, partially localizing with another LEE-encoded effector protein, Tir. EspG was found to interact with mammalian tubulin in both genetic screens and gel overlay assays. Binding to tubulin by EspG caused localized microtubule depolymerization, resulting in actin stress fiber formation through an undefined mechanism. Heterologous expression of EspG in yeast resulted in loss of cytoplasmic microtubule structure and function, preventing coordination between bud development and nuclear division. Yeast expressing EspG were also unable to control cortical actin polarity. We suggest that EspG contributes to the ability of A/E pathogens to establish infection through a modulation of the host cytoskeleton involving transient microtubule destruction and actin polymerization in a manner akin to the Shigella flexneri VirA protein.