We examined whether human sodium/iodide symporter (hNIS) radioiodine gene therapy can modulate the phenotype of cancer cells and enhance the killing activities of CTLs in a mouse tumor model. Various doses of I-131 (75, 300, 600, 1,200, and 2,400 microCi/5 mL) were incubated with hNIS-expressing colon cancer (CT26/hNIS) and parental cells (CT26), and numbers of MHC class I and Fas-expressing cells were determined by fluorescence-activated cell sorting (FACS). In addition, CT26/hNIS or CT26 tumor-bearing mice were treated with 1,200 microCi of I-131, and percentages of MHC class I and Fas-expressing tumor cells were determined by FACS. The levels of tumor-infiltrating CD8+IFNgamma+ and CD11c+CD86+ cells and CTL killing activities were measured in CT26/hNIS tumor-bearing mice (treated with PBS or 1,200 microCi of I-131) by FACS and lactate dehydrogenase assay, respectively. MHC class I and Fas gene expressions were markedly upregulated in CT26/hNIS cells, but not in CT26 cells, in an I-131 dose-dependent manner. The level of MHC class I and Fas-expressing cancer cell were 4.5-fold and 2.1-fold higher in CT26/hNIS tumors than in CT26 tumors, respectively (P < 0.01). Interestingly, numbers of tumor-infiltrating CD8+IFNgamma+ cells and CD11c+CD86+ cells were 5-fold and 2.5-fold higher in I-131-treated tumors than in PBS tumors, respectively (P < 0.001). Furthermore, CTL assays showed significantly more specific tumor cell lysis in I-131 tumors than in PBS tumors (P < 0.01). Our findings suggest that hNIS radioiodine gene therapy can generate tumor-associated immunity in tumor microenvironments and enhance the killing activities of CTLs.