RovA is a transcriptional activator of Yersinia invasin, an outer membrane protein involved in bacterial attachment and invasion across the intestinal epithelium. In Y. enterocolitica, a rovA mutant is attenuated for virulence compared with either wild-type or inv mutant strains, indicating that RovA may regulate additional virulence factors. Here, we used microarray analysis to define the RovA regulon. Curiously, there was little overlap between the RovA regulons of Y. enterocolitica and Y. pestis despite the fact that RovA itself is highly conserved between the two species. Some of these differences are explained by the observation that a number of RovA-regulated loci in Y. enterocolitica do not have orthologues in Y. pestis and vice versa, suggesting that RovA established regulatory control over genetic material acquired after the divergence of the species. Electromobility shift assays demonstrated that 15 of these RovA-regulated loci directly interact with RovA, and 11 of these promoters had similar affinity as observed for the inv promoter. H-NS and YmoA are believed to form a transcriptional repression complex on the inv promoter, and several studies indicate that RovA and H-NS have overlapping DNA binding sites. H-NS and YmoA regulated a subset of the RovA-regulated loci. Furthermore, H-NS directly bound to 14 of the 15 promoters bound by RovA. From these data, we hypothesize that RovA generally behaves as an anti-H-NS factor to alleviate transcriptional repression in Y. enterocolitica. A number of recent studies have presented data and a model suggesting that H-NS functions as a transcriptional silencer of horizontally acquired genes. This repression can be selectively relieved by regulators such as RovA, and the observation that nearly all RovA-activated genes are repressed by H-NS is consistent with this model.