BACKGROUND: Phosphatidylserine (PS) exposed on apoptotic cells promotes immune clearance of dead cells without inducing inflammation. Conversely, PS exposure on live tumor cells promotes an immunosuppressive tumor microenvironment that hinders antitumor immune responses. After confirming elevated PS levels in various tumor cell lines and cancer tissues, we aimed to investigate its potential as a target antigen for chimeric antigen receptor T cell (CAR-T) therapy. METHODS: We used two different approaches to target PS. First, we employed the adaptor proteins, EDAnnexin or BCMAnnexin comprising annexin V and EDA (extra domain A of fibronectin) or B-cell maturation antigen (BCMA) antigens, to redirect the lytic activity of EDA CAR-T or BCMA CAR-T cells toward PS-expressing tumor cells. In a second approach, we developed an annexin V-based CAR (Anxa CAR-T) to directly recognize PS-positive tumor cells. RESULTS: The adaptors proteins EDAnnexin and BCMAnnexin successfully redirected EDA CAR-T or BCMA CAR-T cell activity, leading to an efficient recognition of PS CONCLUSIONS: PS holds promise as a target antigen for CAR-T cell therapy, underscoring the need to address fratricide as a key challenge in the development of PS-targeting CAR-T cells.