CAR-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 against solid tumours. Cancer stem cells are a small population of cells within a tumour that have 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 drives relapse. Targeting cancer stem cells will reduce the tumours’ ability to generate new cells, potentially resulting in durable tumour elimination. We have tested a novel CAR-T 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. LGR5 is a modulator of Wnt/β-catenin signalling, and in cancer LGR5 either potentiates or suppresses Wnt signalling, providing positive selection pressure on cancer cells. To determine whether LGR5 is a suitable tumour target, we have generated six different CAR-T constructs and generated CAR-T cells against LGR5 using lentiviral gene delivery. We find that our manufacturing protocol generates self-renewing naïve-like and central memory LGR5-targeting CAR-T cells, which have the potential to provide long-term protection against tumour growth. We demonstrate that four out of the six LGR5- targeted CAR-T cells significantly kill colorectal and neuroblastoma cell lines in vitro. We tested the top four binders in pilot in vivo experiments and demonstrate the ability of LGR5-targeting CAR-T cells to significantly inhibit the growth of human colorectal tumours in NSG xenograft models. Our data positions LGR5-targeting CAR-T therapy as a viable therapeutic for human metastatic colorectal cancer types, which we aim to test in human clinical trials.