Abstract: Mesothelin is a potential CAR-T target for treating triple-negative breast cancer (TNBC). The majority of Mesothelin CAR-T cells currently developed in clinical trials used scFv derived from murine monoclonal antibody SS1 as antigen binding domain. The murine scFv is prone to induce the production of anti-CAR antibody in the body, which leads to unsatisfactory effect. Therefore, it is urgent to develop new antigen binding domains for constructing CAR-T. A Mesothelin-targeting nanobody VHH with a similar affinity to SS1 scFv was selected. Two CAR-T cells with different antigen recognition domains (scFv and VHH) targeting Mesothelin were constructed, and their killing ability against MDA-MB-231 cells over-expressing Mesothelin was compared. The results showed that VHH CAR-T demonstrated better killing ability against triple negative breast cancer cells than scFv CAR-T cells, which was widely used in clinical trials.In addition, VHH CAR-T expressed more CD107a and CD69 than scFv CAR-T. This is the first Mesothelin CAR-T derived from VHH sequence worldwide. The study provides basis for the further development of Mesothelin CAR-T.