Interleukin-18 has been proposed for cancer immunotherapy for a long time. However, the presence of IL-18 binding protein (IL-18BP) and the unstable form of IL-18 caused the low impact of this protein in human clinical trials. To overcome this, we performed the mutagenesis targeting surface cysteines (C38, C68, C76, and C127) on our modified IL-18 to prevent intermolecular disulfide bond formation. The ORF of wild-type, IL-18 DM and IL-18 DM1234 were synthesized and expressed in E. coli. All IL-18 were refolded by step-wise dialysis and tested for protein aggregation by ProteoStat protein aggregation assay. All recombinant IL-18 were also investigated for activity by IFN-γ induction assay. The structure of modified IL-18 was visualized by molecular dynamic (MD) simulation. The results showed that IL-18 DM1234 demonstrated the lowest aggregation signal compared to others. This protein also displayed higher activity than wild-type and IL-18 DM about 10 and 2.8 times, respectively. MD simulation revealed the binding site I of IL-18 DM1234 was the part mostly affected by C-to-S substitution. In conclusion, this is the first report for IL-18 that the modifications by mutagenesis improved both activity and stability and this IL-18 may be used for medical purposes in the future.