Nucleoside diphosphate kinases (NDPK-C) belong to the NM23 protein family and catalyze the reversible phosphorylation of nucleoside diphosphates to nucleoside triphosphates in a magnesium-dependent manner. The enzymes exist as homo- or heteromeric hexamers, the latter are formed with other isoforms. We described a 1.4 resolution structure of human homo-hexameric NDPK-C bound to ADP. We also analyzed the structure of NDPK-C in the presence of GDP, UDP, and cAMP and described the high-resolution structures of the as-yet unidentified complexes. Both of the partial reactions of the NDPK phosphotransferase activity require Mg2+ ions. We provided the structural basis by comparing NDPK-C nucleotide complexes in the presence and absence of this ion. To investigate the nucleotide impact on the NDPK-C conformation, we analyzed a nucleotide-depleted NDPK-C structure and compared it with the nucleotide bound form. The analysis revealed a conformational change upon substrate binding and allowed us to identify flexible regions in the substrate binding site.