Chimeric antigen receptor T cells have demonstrated robust clinical activity against B-cell malignancies, and rapid translation to approval for use in man has inspired the search for CAR-T cells against solid tumours. Cancer stem cells are a small population of cells within a tumour that has the capacity to self-renew, differentiate, and initiate new tumours. Cancer stem cells are known to be resistant to chemotherapy and radiotherapy and are enriched in residual disease which allows relapse. Targeting cancer stem cells will reduce the tumours’ ability to generate new cells resulting in tumour degeneration. We have tested a novel CAR-T cell that has the potential to target cancer stem cells. Leucine-rich G protein-coupled receptor 5 (LGR5) expression is restricted to stem cell populations in multiple tissues in adults. In addition, LGR5 is a marker of cancer stem cells and LGR5+ cells are implicated in tumour, progression, and metastasis. We have designed CAR-T constructs and generated human CAR-T cells against LGR5 using lentiviral gene delivery. LGR5-targeting CAR-T cells showed significant antigen-specific cytotoxicity against colorectal cancer cell lines in vitro. In preclinical colorectal cancer xenograft mouse model, LGR5-targeting CAR-T cells were able to significantly inhibit the growth of human colorectal tumours with no detectable off-target toxicity. Furthermore, we find that self-renewing naïve-like and central memory LGR5-targeting CAR-T cells provide long-term protection against tumour growth. Our data positions LGR5-targeting CAR-T therapy as a viable therapeutic for human metastatic colorectal cancer types with minimal off-target effects, which we aim to test in human clinical trials.