While Chimeric Antigen Receptor (CAR)-T cells have demonstrated significant efficacy in the treatment of haematological malignancies, there are many challenges to overcome in developing CAR-T cell therapies against solid tumours. In particular, the scarcity of recognised effective target antigens in solid tumours remains a significant hurdle. Leucine-rich repeat-containing G-protein coupled receptor (LGR5) is a cancer stem cell marker, which mediates important roles in tumor initiation and metastasis. Upregulated LGR5 expression in malignant cell contexts highlights this surface receptor as a promising target for CAR-T cell therapy. We produced a library of CARs targeting LGR5 and tested them against a range of human cancers. We demonstrate that LGR5 is expressed, both in vitro and in vivo, by numerous human cancers, including colorectal, ovarian, hepatic, brain, and pancreatic cancer. LGR5-targeting CAR-T cells effectively kill LGR5-expressing cancer cells in in vitro cytotoxicity assays and display significant anti-tumor activity in vivo when administered at 1, 2 or 3 weeks post-tumour inoculation in a subcutaneous human colorectal cancer xenograft mouse model. Furthermore, LGR5-targeting CAR-T cells that induce primary tumour rejection confer complete protection against a secondary tumour challenge 5 weeks post-T cell transfer. In vivo spatiotemporal distribution and repeated dosing studies demonstrate that LGR5-targeting CAR-T cells do not persist in off-target organs by d28 post-tumour inoculation, and they are safe and well-tolerated under the conditions tested. In contrast, these cells accumulate at the tumour site until tumour elimination. These studies allowed selection of a clinical candidate that is currently slated for Phase I/2a clinical trial in patients with advanced colorectal cancer.