〔Abstract〕 Objective To investigate whether dual-receptor chimeric antigen receptor T(CART) cells targeting claudin-18 isoform 2(CLDN18.2) and human epidermal growth factor receptor 2(HER2)can reduce the off-target effect and inhibit the growth of gastric cancer cells. Methods Western blot and flow cytometry were used to detect the expression of CLDN18.2 and HER2 in tumor cells. Tumor cell lines overexpressing CLDN18.2 and HER2 were constructed by lentivirus infection. CART cells were constructed by lentivirus infection. Flow cytometry was used to detect the positive rate and subtype distribution of CART cells. Lactic dehydrogenase(LDH) assay was used to detect the cytotoxicity of CART cells against target tumor cells. The tumor suppressive efficiency of CART cellsin vivowas measured using nude mouse xenograft model. Immunohistochemistry was used to detect the infiltration of CD3+T cells in tumor tissues. Results Gastric cancer cell lines with high or no expression of CLDN18.2 and HER2 were successfully screened, and gastric cancer cell lines with single or simultaneous expression of CLDN18.2 and HER2 were constructed. CART cells Cz, Hbb and CHbbz were successfully constructed. Both Cz and CHbbz had specific dose-dependent cytotoxicity against CLDN18.2+and CLDN18.2+HER2+tumor cell linesin vitro(P<0.001). In the nude mouse transplanted tumor model, only CHbbz could produce significant tumor suppressor activity and T cell tumor infiltration against CH-AGS tumor cells(P<0.001), while only CHbbz could produce significant tumor suppressor activity and T cell tumor infiltration against CLDN18.2+HER2+tumor cells(P<0.001). Conclusion Dual receptor CART cells targeting CLDN18.2 and HER2 can reduce the probability of off-target toxicity and inhibit the growth of gastric cancer, which is a potential therapeutic strategy for gastric cancer.