In Escherichia coli, cytosine DNA methylation is catalyzed by the Dcm (DNA cytosine methylase) protein and occurs at the second cytosine in the sequence 5′CCWGG3′. Although the presence of cytosine DNA methylation was reported over 35 years ago, the biological role of 5‐methylcytosine in E. coli remains unclear. There is data indicating that Dcm can protect the genome against attack by a restriction enzyme that cleaves the same sequence, yet DNA methyltransferases in eukaryotes often influence gene expression. In order to gain insight into the potential roles of cytosine DNA methylation in E. coli, we: (a) screened 162 strains including laboratory strains, pathogens, and recently isolated environmental samples for the presence of the full‐length dcm gene using the polymerase chain reaction; (b) examined the same strains for the presence of 5‐methylcytosine at 5′CCWGG3′ sites using a restriction enzyme isoschizomer digestion assay; and (c) quantified the levels of 5‐methyl‐2′‐deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm‐mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5‐methylcytosine ranges from 0.8–1.2% of the cytosines. We also tested the hypothesis that Dcm influences gene expression. Gene expression in wild‐type and dcm knockout cells was compared via qPCR. We focused on ribosomal protein genes, as they have been previously demonstrated to contain numerous 5′CCWGG3′ sites. We demonstrate that Dcm represses expression of ribosomal protein genes during stationary phase, and this may explain the ubiquitous presence of this DNA modification pathway. Support for this work was provided by the Geneseo Foundation and NIH grant R15AI074035‐01.