Abstract Purpose Currently, approximately 11–38% of prostate cancer (PCa) patients undergoing radical prostatectomy have a positive surgical margin (PSM) on histopathology. Cerenkov luminescence imaging (CLI) using ⁶⁸Ga-prostate-specific membrane antigen (⁶⁸Ga-PSMA) is a novel technique for intraoperative margin assessment. The aim of this first-in-man study was to investigate the feasibility of intraoperative ⁶⁸Ga-PSMA CLI. In this study, feasibility was defined as the ability to distinguish between a positive and negative surgical margin, imaging within 45 min and low radiation exposure to staff. Methods Six patients were included in this ongoing study. Following perioperative i.v. injection of ~ 100 MBq ⁶⁸Ga-PSMA, the prostate was excised and immediately imaged ex vivo. Different acquisition protocols were tested, and hotspots on CLI images from the intact prostate were marked for comparison with histopathology. Results By using an acquisition protocol with 150 s exposure time, 8 × 8 binning and a 550 nm shortpass filter, PSMs and negative surgical margins (NSMs) were visually correctly identified on CLI in 3 of the 5 patients. Two patients had a hotspot on CLI from cancer < 0.1 mm from the excision margin. Conclusion Overall, the study showed that ⁶⁸Ga-PSMA CLI is a feasible and low-risk technique for intraoperative margin assessment in PCa. The remaining patients in this ongoing study will be used to assess the diagnostic accuracy of the technique. Trial registration: NL8256 registered at www.trialregister.nl on 04/11/20109.