Major human Nucleoside diphosphate kinases (NDPKs) exist as hetero-oligomers, consisting of NDPK-A and NDPK-B, rather than homo-oligomer. To investigate their biological function depending on the oligomeric structure in vivo, we characterized the biochemical properties of cellular NDPK. Cellular NDPKs, which are made up of a unique combination of isoforms, were purified from human erythrocyte and placenta. We found that cellular NDPK and recombinant isoforms NDPKs have their own distinct biochemical properties in autophosphorylation, stability toward heat or urea, and DNA binding. Cellular NDPK was found to have unique characteristics rather than the expected additive properties of recombinant isoforms. The mutations in the dimeric interface of NDPK-B (R34G, N69H or K135L) caused defective DNA binding and simultaneously reduced the enzymatic stability These results suggest that the oligomeric interaction could play a major role in the stability of catalytic domain and might be related to the regulation of various cellular functions of NDPK.