Atypical enteropathogenic Escherichia coli (aEPEC) causes endemic diarrhea, diarrheal outbreaks, and persistent diarrhea in humans, but the mechanism by which aEPEC causes disease is incompletely understood. Virulence regulators and their associated regulons, which often include adhesins, play key roles in the expression of virulence factors in enteric pathogenic bacteria. In this study we identified a transcriptional regulator, RalR, in the rabbit-specific aEPEC strain, E22 (O103:H2) and examined its involvement in the regulation of virulence. Microarray analysis and quantitative real-time reverse transcription-PCR demonstrated that RalR enhances the expression of a number of genes encoding virulence-associated factors, including the Ral fimbria, the Aap dispersin, and its associated transport system, and downregulates several housekeeping genes, including fliC. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins and by adherence and motility assays. To investigate the mechanism of RalR-mediated activation, we focused on its most highly upregulated target operons, ralCDEFGHI and aap. By using primer extension, electrophoretic mobility shift assay, and mutational analysis, we identified the promoter and operator sequences for these two operons. By employing promoter-lacZ reporter systems, we demonstrated that RalR activates the expression of its target genes by binding to one or more 8-bp palindromic sequences (with the consensus of TGTGCACA) located immediately upstream of the promoter core regions. Importantly, we also demonstrated that RalR is essential for virulence since infection of rabbits with E22 carrying a knockout mutation in the ralR gene completely abolished its ability to cause disease.