We performed whole genome analyses of DNA methylation in Shewanella oneidensis MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-Molecule Real Time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for Type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found S. oneidensis MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes in these conditions, suggesting DNA methylation is not a major regulator of gene expression in S. oneidensis MR-1. The enrichment of methylated GATC motifs in the origin of replication indicate DNA methylation may regulate genome replication in a manner similar to that seen in Escherichia coli. Furthermore, comparative analyses suggest that many Gammaproteobacteria, including all members of the Shewanellaceae family, may also utilize DNA methylation to regulate genome replication.