Development of specific immunotherapy for colorectal cancer (CRC) will require identification of antigens selectively or exclusively expressed on CRC cells and strategies to induce and enhance immune responses against these antigenic targets. Cancer-testis (C-T) antigens are proving to be excellent targets for immunotherapy of solid tumors such as melanoma, but their clinical utility for treatment of CRC has to date been limited by their infrequent expression in CRC cells. Here we report that the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) induces expression of NY-ESO-1 and other C-T genes in CRC cells both in vitro and in vivo in a dose-dependent manner but has negligible effects on the expression of C-T genes in normal nontransformed cells such as fibroblasts. The induction by DAC of NY-ESO-1 expression in CRC cells persists over 100 days after DAC exposure and is associated with increased levels of NY-ESO-1 protein. CRC cells exposed to DAC at concentrations that can be readily achieved in vivo are rendered susceptible to major histocompatibility complex-restricted recognition by CD8 NY-ESO-1-specific T cells. We also demonstrate that retroviral transduction of polyclonal peripheral blood T cells from a metastatic CRC patient with the T-cell receptor α-chain and β-chain genes encoding a human leukocyte antigen-A2-restricted, NY-ESO-1157-165-specific T-cell receptor can be used to generate both CD8 and CD4 NY-ESO-1157-165-specific T cells that selectively recognize DAC-treated CRC but not nontransformed cells. Collectively, these results suggest that the combination of epigenetic modulation and adoptive transfer of genetically engineered T lymphocytes may enable specific immunotherapy for CRC.