<i>Citrobacter rodentium</i> utilizes a type 3 secretion system (T3SS) to inject effector proteins into host intestinal epithelial cells, causing structural and functional changes in these cells during infection. Here, we examined the effects of <i>C. rodentium </i>infection on host cell protein ubiquitination in vivo. We observed the appearance of ubiquitinated protein (Ub⁺) aggregates in intestinal epithelial cells near the site of bacterial attachment. Formation of aggregates was dependent on T3SS activity and the effector translocated intimin receptor (Tir). Aggregates formed at 6 days after infection, when bacterial loads were maximal, but were absent at 12 days. Aggregates were not observed in MyD88^–/– mice. Aggregate formation correlated with MyD88-dependent induction of NADPH oxidase 1, implicating reactive oxygen species in their formation. Aggregates were also observed in gastric tissues of mice infected with <i>Helicobacter pylori.</i> This is the first report describing the formation of Ub⁺ aggregates in vivo during enteric infection, and reveals that this phenotype is dependent on both bacterial and host factors. Our experiments extend previous in vitro studies suggesting that Ub⁺ aggregates play an important role in the initiation of immune responses to infection. Ub⁺ aggregates are a novel marker of the cellular response to enteric pathogens and will be useful for studies of host-pathogen interactions in vivo.