Helicobacter pylori is a worldwide pathogen currently estimated to be actively residing in about half of the world’s population. H. pylori strains are remarkable for the number of DNA methyltransferases encoded in their genomes, and it is becoming increasingly evident that DNA methylation has an important role in the gene expression. In this study, I characterized a C-5 cytosine DNA methyltransferase (M.Hpy99III) that is conserved in H. pylori strains and converts GCGC sites to Gm5CGC. A M.Hpy99III-deficient mutant in H. pylori G27 displayed growth and motility defects. A strong positional bias for GCGC motifs in the -13 region of H. pylori promoter was observed. Expression of green fluorescent protein (gfp) reporter genes constructed with selected GCGC-containing promoters indicated activities of some of these promoters was reduced in the M.Hpy99III-deficient mutant, which indicated many of the GCGC-containing promoters in H. pylori may be regulated by DNA methylation.