Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has made significant success in treating hematological malignancies, paving the way for solid tumors like prostate cancer. However, progress is impeded by a paucity of suitable target antigens. A novel carbohydrate antigen, F77, is expressed on both androgen-dependent and androgen-independent prostate cancer cells, making it a potential immunotherapy target. This study entails the generation and evaluation of a second-generation CAR against a carbohydrate antigen on malignant prostate cancer cells. Using a single chain fragment variable (scFv) from an F77-specific mouse monoclonal antibody, we created second-generation CARs with CD28 and CD137 (4-1BB) costimulatory signals. F77 expressing lentiviral CAR T cells produce cytokines and kill tumor cells in a F77 expression-dependent manner. These F77-specific CAR T cells eradicate prostate tumors in a human xenograft model employing PC3 cells. These findings validate F77 as a promising immunotherapeutic target for prostate cancer and other malignancies with this aberrant carbohydrate structure.Keywords: CAR T therapy; F77; Glycan therapy; Prostate cancer.