The ptsHI operon from Leuconostoc mesenteroides ssp. mesenteroides SY1 (L. mesenteroides SY1), a strain isolated from kimchi, was cloned and characterized. The ptsH open reading frame (ORF) was 273 bp in size, which can encode a protein of 90 amino acid residues with a molecular weight of 9,212 Da. The pfsI ORF was 1,719 bp in size, which was capable of encoding a protein of 572 amino acids with a molecular mass of 62,549 Da. ptsH and pfsI genes were transcribed as a single transcript of 2.0 kb in size regardless of carbon sources, supporting the operon structure. Although the deduced amino acid sequences of the HPr and EI were highly homologous with those of other Gram-positive bacteria, an additional amino acid (glutamine at the $3^{rd}$ amino acid) was present in HPr from L. mesenteroides SY1. Phosphorylation sites of HPr included the histidine residue ($16^{th}$) and serine residue ($47^{th}$). Mutant HPrs, in which each phosphorylation site was mutated into alanine, were obtained, and phosphorylation with HPr and mutated HPrs showed that HPr was phosphorylated at the serine residue ($47^{th}$) by HPr kinaseiphosphorylase (HPr K/P).