Carnosine dipeptidase II (CN2/CNDP2) is an M20 family metallopeptidase that hydrolyses various dipeptides including β-alanyl-l-histidine (carnosine). Crystallographic analysis showed that CN2 monomer is composed of one catalytic and one dimerization domains, and likely to form homodimer. In this crystal, H228 residue of the dimerization domain interacts with the substrate analogue bestatin on the active site of the dimer counterpart, indicating that H228 is involved in enzymatic reaction. In the present study, the role of intradimer interaction of CN2 in its catalytic activity was investigated using electrospray-ionization time-of-flight mass spectrometry (ESI-TOF MS). First, a dimer interface mutant I319K was prepared and shown to be present as a folded monomer in solution as examined by using ESI-TOF MS. Since the mutant was inactive, it was suggested that dimer formation is essential to its enzymatic activity. Next, we prepared H228A and D132A mutant proteins with different N-terminal extended sequences, which enabled us to monitor dimer exchange reaction by ESI-TOF MS. The D132A mutant is a metal ligand mutant and also inactive. But the activity was partially recovered time-dependently when H228A and D132A mutant proteins were incubated together. In parallel, H228A/D132A heterodimer was formed as detected by ESI-TOF MS, indicating that interaction of a catalytic center with H228 residue of the other subunit is essential to the enzymatic reaction. These results provide evidence showing that intradimer interaction of H228 with the reaction center of the dimer counterpart is essential to the enzymatic activity of CN2.