The prostate specific membrane antigen (PSMA) is broadly overexpressed on prostate cancer (PCa) cell surfaces. In this study, we report the synthesis, characterization, in vitro binding assay, and in vivo magnetic resonance imaging (MRI) evaluation of PSMA targeting superparamagnetic iron oxide nanoparticles (SPIONs). PSMA-targeting polypeptide CQKHHNYLC was conjugated to SPIONs to form PSMA-targeting molecular MRI contrast agents. In vitro studies demonstrated specific uptake of polypeptide-SPIONs by PSMA expressing cells. In vivo MRI studies found that MRI signals in PSMA-expressing tumors could be specifically enhanced with polypeptide-SPION, and further Prussian blue staining showed heterogeneous deposition of SPIONs in the tumor tissues. Taken altogether, we have developed PSMA-targeting polypeptide-SPIONs that could specifically enhance MRI signal in tumor-bearing mice, which might provide a new strategy for the molecular imaging of PCa.