The Nm23-H1 gene is a metastasis suppressor gene. However, its biochemical mechanism of suppressing the metastatic potential of cancer cells is still unknown. The previous hypothesis that a histidine protein kinase activity may contributes to the motility-suppressive effect of Nm23-H1 could not explain why the H118F mutant, a kinase-deficient mutant, still had motility-suppressive ability. We conducted a study on the double mutant P96S/S120G of Nm23-H1 and succeeded in introducing the RP-HPLC method in NDPK assay. The results showed that the double mutant P96S/S120G, when expressed in the bacteria, was completely aggregated in inclusion bodies; this mutant abrogated not only its motility-suppressive ability, but also its NDPK activity. Based on previous work and this study, we prompted that the deficiency of motility-suppressive function of S120G, P96S, and P96S/S120G mutants was due to their altered structure, which might deprive Nm23-H1 of most activities including kinase activity or interactions with other proteins.