Acinetobacter baumannii is a new threat in intensive care units (ICUs) for its multiresistance to antibiotics, but little is known about this bacterium. Nucleoside diphosphate kinase (NDK) is an evolutionarily conserved enzyme that catalyzes phosphoryl transformation between nucleosides. In our study, the crystal structure of wild type Acinetobacter baumannii NDK along with its mutant generated through truncation of the C-terminal arginine-threonine-arginine (RTR) residues, were solved. In comparison with Myxococcus xanthus NDK structure, we speculated that Acinetobacter baumannii NDK shared a similar catalytic mechanism with Myxococcus xanthus. Activity assay and CD spectra analysis revealed that E28A mutant might interrupt the secondary structure of the protein leading to declined enzymatic activity. Truncation of the C-terminal RTR residues would lead to the instability of the tertiary structure resulting in reduced kinase activity. Lys33 was a key residue for maintaining dimer interaction when RTR residues were truncated but was not sufficient to keep efficient enzymatic reaction. The structural data can provide a potential target to develop novel therapeutic approaches to overcome multiresistance of the bacterium against antibiotics.