Epigenetic DNA methylation modulates several important bacterial cellular processes, such as mismatch repair, gene regulation, transposition and chromosome replication. Restriction Modification Systems (RMSs) utilise DNA methylation to distinguish between self and non-self DNA but, this epigenetic mark has the potential to alter gene expression and by extension virulence. The objective of this study was to systematically elucidate the role of Type I RMS mediated DNA methylation in Escherichia coli virulence and general physiology. SMRT sequencing was used to identify a novel Type I RMS mediated DNA methylation motif in prototypic Uropathogenic E. coli (UPEC) cystitis isolate UTI89. Deletion or replacement of Type I methylation in UPEC strain UTI89 does not have any effect in specific in vitro assays, such as growth rate, motility, biofilm formation and hemagglutination. This trend continues in far more sensitive assays such as transurethral in vivo infection, RNA-seq and Phenotype Microarray, with no effect observed for Type I DNA methylation. Similar results were observed in UPEC strain CFT073 and non-pathogenic E. coli K12 substrain MG1655. To summarise, our systematic scrutiny revealed that Type I DNA methylation has no effect on any phenotypic traits in three different E. coli strains with distinct and functional Type I RMSs, under the conditions tested. This is in contrast with published studies which describe global gene expression changes as a consequence of altered DNA methylation.