Prostate-specific Membrane Antigen (PSMA) is overexpressed in prostate cancer (PCa) and a promising target for molecular imaging and therapy. Nanobodies (single domain antibodies, VHH) are the smallest antibody-based fragments possessing ideal molecular imaging properties, such as high target specificity and rapid background clearance. We developed a novel anti-PSMA nanobody (JVZ-007) for targeted imaging and therapy of PCa. Here, we report on the application of the (111)In-radiolabeled nanobody for SPECT/CT imaging of PCa.A nanobody library was generated by immunization of a llama with four human PCa cell lines. Anti-PSMA nanobodies were captured by biopanning on PSMA overexpressing cells. JVZ-007 was selected for evaluation as imaging probe. JVZ-007 was initially produced with a c-myc-hexahistidine (his) tag allowing purification and detection. The c-myc-his tag was subsequently replaced by a single cysteine at the C-terminus, allowing site-specific conjugation of chelates for radiolabeling. JVZ-007-c-myc-his was conjugated to p-SCN-DTPA via the lysines, while JVZ-007-cys was conjugated to maleimide-DTPA via the C-terminal cysteine. PSMA-targeting was analyzed in vitro by cell binding experiments using flow cytometry, autoradiography and internalization assays using various PCa cell lines and patient-derived xenografts (PDXs). Targeting properties of radiolabeled nanobodies were evaluated in vivo in biodistribution and SPECT/CT imaging experiments, using nude mice bearing PSMA-positive PC-310 and PSMA-negative PC-3 tumors.JVZ-007 was successfully conjugated to DTPA for radiolabeling with (111)In at room temperature. (111)In-JVZ007-c-myc-his and (111)In-JVZ007-cys internalized in LNCaP cells, and bound to PSMA-expressing PDXs, and importantly not to PSMA-negative PDXs and human kidneys. Good tumor targeting and fast blood clearance were observed for (111)In-JVZ-007-c-myc-his and (111)In-JVZ-007-cys. Renal uptake of (111)In-JVZ-007-c-myc-his was initially high, but was efficiently reduced by co-injection of gelofusine and lysine. The replacement of the c-myc-his tag by the cysteine contributed to further reduction of renal uptake without loss of targeting. PC-310 tumors were clearly visualized by SPECT/CT with both tracers, with very low renal uptake (< 4\%ID/g) for (111)In-JVZ-007-cys already at 3 h after injection.We developed an (111)In-radiolabeled anti-PSMA nanobody, showing very good tumor targeting, low uptake in non-target tissues, and low renal retention, allowing excellent SPECT/CT imaging of PCa within a few hours after injection.