EcoHK31I DNA methyltransferase recognizes the sequence 5′-YGGCCR-3′ and adds a methyl group to the fifth position of the internal cytosine to protect the DNA from cleavage by its cognate endonuclease. M.EcoHK31I is composed of polypeptides α and β. Polypeptide β only contains the conserved IX motif of the C5-MTase family, and provides a unique example to show that this motif alone may be dislocated to another polypeptide. By electromobility shift assay, we found that the α/β complex recognizes specific oligonucleotide substrates. Polypeptide β formed aggregates with DNA, while polypeptide β alone did not bind DNA. Therefore, polypeptide β assists in the proper binding of polypeptide β to DNA substrate. The complex of polypeptide α and a polypeptide β variant with an N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive methyltransferase. The dissociation equilibrium constant (Kd) of the α/β complex was 56.4 nM, while the Kd value for the α/ΔN46-polypeptide β complex was increased approximately 95-fold, caused by a drastic decrease in dissociate rate constant (kd) and an increase in the association rate constant (ka). This indicates that the N-terminal region of polypeptide β takes part in subunit interaction, while the C-terminal region is involved in DNA binding.