Monoclonal antibodies against mesothelin are being evaluated for the treatment of mesothelioma and multiple forms of cancers, and show great promise for clinical development for solid cancers. Antibodies against mesothelin have been shown to act via immunotoxin-based inhibition of tumor growth and induction of antibody-dependent cell-mediated cytotoxicity (ADCC). However, complement-dependent cytotoxicity (CDC), considered an important additional mechanism of therapeutic antibodies against tumors, is inactive for such antibodies. Here, we used phage display antibody engineering technology and synthetic peptide screening to identify SD1, a human single-domain antibody to mesothelin. SD1 recognizes a conformational epitope at the C-terminal end (residues 539–588) of mesothelin close to the cell surface. To investigate SD1 as a potential therapeutic agent, we generated a recombinant human Fc (SD1-hFc) fusion protein. Interestingly, the SD1-hFc protein exhibits strong CDC activity, in addition to ADCC, against mesothelin-expressing tumor cells. Furthermore, it causes growth inhibition of human tumor xenografts in nude mice as a single agent. SD1 is the first human single-domain antibody targeting mesothelin-expressing tumors, shows potential as a cancer therapeutic candidate, and may improve current antibody therapy targeting mesothelin-expressing tumors. Mol Cancer Ther; 12(4); 416–26. ©2013 AACR.