Transforming growth factor (TGF)-β is a potent immunosuppressant. Overproduction of TGF-β by tumor cells may lead to tumor evasion from the host immune surveillance and tumor progression. The present study was conducted to develop a treatment strategy through adoptive transfer of tumor-reactive TGF-β-insensitive CD8⁺ T cells. The mouse TRAMP-C2 prostate cancer cells produced large amounts of TGF-β1 and were used as an experimental model. C57BL/6 mice were primed with irradiated TRAMP-C2 cells. CD8⁺ T cells were isolated from the spleen of primed animals, were expanded ex vivo, and were rendered TGF-β insensitive by infecting with a retrovirus containing dominant-negative TGF-β type II receptor. Results of in vitro cytotoxic assay revealed that these CD8⁺ T cells showed a specific and robust tumor-killing activity against TRAMP-C2 cells but were ineffective against an irrelevant tumor line, B16-F10. To determine the in vivo antitumor activity, recipient mice were challenged with a single injection of TRAMP-C2 cells for a period up to 21 days before adoptive transfer of CD8⁺ T cells was done. Pulmonary metastasis was either eliminated or significantly reduced in the group receiving adoptive transfer of tumor-reactive TGF-β-insensitive CD8⁺ T cells. Results of immunofluorescent studies showed that only tumor-reactive TGF-β-insensitive CD8⁺ T cells were able to infiltrate into the tumor and mediate apoptosis in tumor cells. Furthermore, transferred tumor-reactive TGF-β-insensitive CD8⁺ T cells were able to persist in tumor-bearing hosts but declined in tumor-free animals. These results suggest that adoptive transfer of tumor-reactive TGF-β-insensitive CD8⁺ T cells may warrant consideration for cancer therapy.