Small molecule ligands targeting human glutamate carboxypeptidase II (GCPII) are used in diverse diagnostic and therapeutic applications ranging from prostate cancer imaging to the therapy of neurological disorders. Such inhibitors typically consist of a glutamate moiety linked to a zinc-binding group to ensure high specificity and affinity for GCPII, respectively. At present, there are no structural data describing interactions between GCPII and inhibitors harboring a hydroxamate function, the prominent zinc-binding function used in the field. Here we report X-ray structures of several complexes between GCPII and hydroxamate-based inhibitors. Our structures reveal unexpected positioning of hydroxamates in the internal GCPII pocket that differs markedly from binding modes of matching prototypical GCPII inhibitors featuring different zinc-binding groups. The data can be exploited for the structure-assisted design of novel GCPII-specific inhibitors.