Succinate upregulates enterohaemorrhagic Escherichia coli (EHEC) virulence. Lysine succinylation, a post-translational modification, regulates cellular function in eukaryotes but is less characterized in bacteria. We hypothesized that lysine succinylation regulates EHEC virulence. Here we used SILAC-based proteomics and characterized the EHEC succinylome to show that the transcription factor, PurR, is succinylated at K24 and K55. Succinylation of PurR inhibited its ability to directly bind DNA and repress expression of a major virulence factor, the Type 3 Secretion System (T3SS), thus increasing T3SS expression. Deletion of purR, or K24E or K55E mutation, increased EHEC adherence to cells and colonization of infant rabbits. Using mice treated with streptomycin to deplete succinate, or colonized with succinate-producing Prevotella copri to increase succinate levels, we showed that microbiota-derived succinate increased succinylation of PurR to promote virulence of Citrobacter rodentium, a model for EHEC, in mice. Lastly, we identified CitC as the succinyltransferase required for PurR modification.