Erwinia carotovora subsp. carotovora is a causal agent of soft-rot diseases in a wide variety of plants. Here, we have isolated a new regulatory factor involved in the virulence of E. carotovora subsp. carotovora by in vivo insertional mutagenesis using a transposon Tn5. The gene was homologous to cytR encoding a transcriptional repressor of nucleoside uptake and catabolism genes in Escherichia coli, Salmonella typhimurium, and Vibrio cholerae. Phenotypic characterization of a nonpolar deletion mutant of the cytR homologue (ΔcytR) revealed that the ΔcytR mutant produced a reduced level of polygalacturonase (Peh) and lost its motility compared to that in the parental strain. With electron microscopy, the ΔcytR mutant was shown to be aflagellate. Furthermore, the expression of fliA and fliC (encoding σ28 and flagellin, respectively) was also reduced in ΔcytR mutant. The virulence of ΔcytR mutant was reduced in Chinese cabbage and potato compared to that of the parental strain. These results suggest that the CytR homologue of E. carotovora subsp. carotovora positively controls Peh production and flagellum synthesis and plays an important role in its pathogenicity.