Significance Because of its selective expression on the surface of a variety of different cancer cells, but not on their normal counterparts, nucleolin (NCL) represents an attractive target for antineoplastic treatments. However, previously described NCL-targeting molecules, although promising, still suffer from intrinsic limitations. Here, we describe the identification of the first fully human anti-NCL immune-based agent displaying antineoplastic activity against solid tumors, both in vitro and in vivo. This molecule could represent the prototype of a novel class of NCL-targeting drugs with enormous clinical potential as tools for the diagnosis and therapy of a wide range of human cancers.