A small percentage of natural Escherichia coli isolates (both commensal and pathogenic) have a mutator phenotype related to defects in methyl-directed mismatch repair (MR) genes. We investigated whether there was a direct link between the mutator phenotype and virulence by (i) studying the relationships between mutation rate and virulence in a mouse model of extraintestinal virulence for 88 commensal and extraintestinal pathogenic E. coli isolates and (ii) comparing the virulence in mice of MR-deficient and MR-proficient strains that were otherwise isogenic. The results provide no support for the hypothesis that the mutator phenotype has a direct role in virulence or is associated with increased virulence. Most of the natural mutator strains studied displayed an unusual virulence phenotype with (i) a lack of correspondence between the number of virulence determinants and pathogenicity in mice and (ii) an intermediate level of virulence. On a large evolutionary scale, the mutator phenotype may help parasites to achieve an intermediate rate of virulence which mathematical models predict to be selected for during long-term parasite-host interactions.