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 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 allows relapse. Targeting cancer stem cells will reduce the tumours’ ability to generate new cells, resulting in tumour remission. 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 designed six lentiviral CAR-T constructs targeting the extracellar domain of LGR5 with differing linker lengths and binding domain orientation, and tested gene delivery to purified human CD3 T cells and in vitro cytotoxicity. All constructs were expressed robustly (>90%), and four of six LGR5-targeting CAR-T cells showed significant antigen-specific cytotoxicity against colorectal cancer and neuroblastoma cell lines in vitro. We confirmed the on-target specificity of the LGR5 targeting CAR T using a cell line overexpressing the LGR5 protein and antibody competition assays, in which an LGR5 MoAb blocked cytotoxicity. Using optimised gene delivery and CAR-T manufacturing protocols, we then generated pre-clinical scale batches of the four lead constructs to evaluate their potency in a preclinical colorectal cancer NSG mouse xenograft model. Post expansion our cell products expressed markers of self-renewing naïve-like and central memory phenotype and three out of four LGR5-targeting CAR-T cells were able to significantly inhibit the growth of human colorectal tumours. These results suggest a CAR-T targeting LGR5 may 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, which we now aim to test in human clinical trials.