Purpose The authors evaluated whether the cytotoxicity of CD8+CTLs generated by combined hMUC1 vaccination and hNIS radioiodine gene therapy was enhanced in the presence of CpG in an established tumor model. Methods CMNF cells (CT26 cells expressing hMUC1, hNIS and Firefly luciferase) were transplanted into BALB/c mice. Four and 10 days later, tumor-bearing mice were immunized intramuscularly with pcDNA3.1 or pcDNA-hMUC1 or pcDNA-hMUC1+CpG, and subsequently administered PBS or $^{131}I$ [five groups (seven mice/group): referred to as the pcDNA3.1+PBS, phMUC1+PBS, pcDNA3.1+$^{131}I$, phMUC1+$^{131}I$, and phMUC1+$^{131}I$+CpG groups]. The number of CD8+IFNr+ T cells of splenocytes as well as the number of CD8+IFNr+ T cells of splenocytes re-stimulated with CD11c+ cells was determined using FACS analysis. The activities of cytotoxic T cells (CTLs) from splenocytes were investigated. Results Marked tumor growth inhibition was observed in the phMUC1+$^{131}I$ and phMUC1+$^{131}I$+CpG groups, but not in the other three single therapy groups. Particularly the number of CD8+IFN-${gamma}$+ T cells of splenocytes was more increased in the phMUC1+$^{131}I$+CpG group than in the phMUC1+$^{131}I$ group. The number of CD8+IFN-${gamma}$+ T cells of splenocytes stimulated with CD11c+ cells was the most enhanced in the phMUC1+$^{131}I$+CpG group among the five groups. Concurrently, the activities of hMUC1-associated CTLs obtained from splenocytes in the phMUC1+$^{131}I$+CpG group were significantly greater than in the other four groups (pcDNA+PBS, phMUC1+PBS, pcDNA+$^{131}I$, phMUC1+$^{131}I$, and phMUC1+$^{131}I$+CpG, $16{pm}2%$, $20{pm}1%$, $30{pm}2%$, $60{pm}2%$, and $87{pm}2%$, respectively, P<0.01). Conclusion Our data suggest that adjuvant CpG ODNs can increase the killing activities of CTLs generated by combined hMUC1 DNA vaccination and hNIS radioiodine gene therapy.