Ovarian cancer is the second leading cause of cancer-related mortality among gynaecological cancers in the Western world. CAR-T cell therapy is an adoptive immunotherapy used to treat some blood cancers. This study has investigated whether leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a suitable target for CAR-T cell therapy for ovarian cancer. We assessed the cytotoxic effects of LGR5-targeting CAR-T cells on a range of ovarian cancer cell lines and primary serous ovarian cancer cells derived from patient ascites (at diagnosis and following relapse with chemotherapy resistant disease) in monolayer culture, 3D spheroid assays and patient-derived explant assays. OVCAR3 and a chemoresistant patient-derived xenograft (PDX) models have been used to assess whether LGR5-targeting CAR-T cells can inhibit growth and metastasis in vivo. We found that LGR5 is highly expressed in high-grade serous ovarian carcinoma (HGSOC) cancer cell lines and LGR5 expression was increased in metastatic and chemotherapy resistant ovarian cancer. We demonstrated that LGR5-targeting CAR-T cells were cytotoxic and significantly inhibited the survival of ovarian cancer cell lines and chemotherapy-resistant primary ovarian cancer cells with high LGR5 expression levels in both monolayer culture and 3D spheroids compared to un-transduced CD3+ T cells. In patient-derived explant assays, we observed increased apoptosis in HGSOC tissues expressing high LGR5 following treatment with LGR5-targeting CAR-T cells. In vivo, LGR5-targeting CAR-T cell treatment decreased the tumour burden in mice with OVCAR3 xenografts and a chemotherapy-resistant PDX model with high LGR5 expression, compared to CD3+ T cells. In summary, LGR5-targeting CAR-T cells have great potential to be developed as a novel immunotherapy for improving ovarian cancer patient outcomes.