Yersinia pestis, the causative agent of plague, is a facultative intracellular pathogen. Previous studies have indicated that the ability of Y. pestis to survive inside macrophages may be critical during the early stages of plague pathogenesis. To gain insights into the biology of intracellular Y. pestis and its environment following phagocytosis, we determined the genome-wide transcriptional profile of Y. pestis KIM5 replicating inside J774.1 macrophage-like cells using DNA microarrays. At 1.5, 4, and 8 h postinfection, a total of 801, 464, and 416 Y. pestis genes were differentially regulated, respectively, compared to the level of gene expression of control bacteria grown in tissue culture medium. A number of stress-response genes, including those involved in detoxification of reactive oxygen species, as well as several metabolic genes involved in macromolecule synthesis, were significantly induced in intracellular Y. pestis, consistent with the presence of oxidative stress and nutrient starvation inside Yersinia-containing vacuoles. A putative stress-induced operon consisting of y2313, y2315, and y2316 (y2313-y2316), and a previously unidentified open reading frame, orfX, was studied further on the basis of its high level of intracellular expression. Mutant strains harboring either deletion, Deltay2313-y2316 or DeltaorfX, exhibited diverse phenotypes, including reduced effector secretion by the type III secretion system, increased intracellular replication, and filamentous morphology of the bacteria growing inside macrophages. The results suggest a possible role for these genes in regulating cell envelope characteristics in the intracellular environment.