Cancer regression by gene-modified T cells bearing a chimeric antigen receptor (CAR) exodomain of mouse origin can be limited by the induction of transgene immunogenicity resulting in poor persistence and function in vivo. The development of functionally-active CAR of human origin can address this issue. Here, we constructed and evaluated fully human anti-mesothelin CARs comprised of a human mesothelin-specific single-chain antibody variable fragment (P4 scFv) coupled to T cell signaling domains. Primary human T cells expressing P4 CAR specifically produced proinflammatory cytokines, degranulated and exerted potent cytolytic functions when cultured with mesothelin-expressing tumors in vitro. P4 CAR T cells also mediated bystander killing of mesothelin-negative cancer cells during coculture. CAR reactivity was not abrogated by soluble tumor-secreted or recombinant mesothelin protein even at supraphysiological levels. Importantly, adoptive transfer of P4 CAR-expressing T cells mediated the regression of large, established tumor in the presence of soluble mesothelin in a xenogenic model of human ovarian cancer. Thus, primary human T cells expressing fully human anti-mesothelin CAR efficiently kill mesothelin- expressing tumors in vitro and in vivo and have the potential to overcome the issue of transgene immunogenicity that may limit CAR T cell trials that utilize scFvs of mouse origin.