Abstract Purpose Mucosal melanoma, a highly aggressive form of skin cancer, remains challenging to manage due to the lack of effective therapies. Mucin 18 (MUC18) is overexpressed in both primary and metastatic lesions of melanoma but rarely in normal tissues. The expression profile makes MUC18 a potential target for development of therapeutic antibodies or chimeric antigen receptor-T (CAR-T) cell therapy. This study aims to generate an effective CAR-T targeting MUC18-positive melanoma and evaluate its preclinical antitumor activity. Experimental design A humanized anti-MUC18 single chain antibody fragment (scFv) was used to construct CAR-T with various designs of the hinge, transmembrane, co-stimulatory, and CD3ζ domains. The antitumor efficacy of MUC18 CAR-T cells was assessed in vitro, in MUC18-positive primary and rechallenged xenograft models, as well as in patient-derived xenograft (PDX) models of human mucosal melanoma. Results The humanized scFv selectively bound to MUC18 with high affinity. Various MUC18 CAR-T cells specifically killed MUC18-positive melanoma cells and could proliferate as a result of exposure to antigen. Among them, CAR-T cells containing an IgG4-derived hinge domain and a CD28 co-stimulatory domain demonstrated superior antitumor efficiency. Robust tumor regression and CAR-T cell expansion were observed in multiple MUC18-positive xenograft models after treatment with the IgG4 hinge and CD28 empowered CAR-T cells. Conclusions This study demonstrated the development of a novel CAR-T therapy for mucosal melanoma, MUC18 CAR-T, that showed strong potency in tumor eradication and inhibition of tumor relapse. This candidate CAR-T therapy could provide a promising strategy for the treatment of the refractory melanoma.