<p>We report a rare case of urothelial carcinoma arising in a horseshoe kidney, successfully treated with robot-assisted nephroureterectomy (RANU) using indocyanine green (ICG) fluorescence imaging to facilitate safe isthmectomy. A 76-year-old male with a left renal pelvic tumor was diagnosed with high-grade urothelial carcinoma (cT1N0M0). Preoperative imaging and three-dimensional reconstruction revealed complex vascular supply from the renal artery, an artery arising near the inferior mesenteric artery, and multiple branches from the common iliac artery, as well as bilateral ureters coursing close to the isthmus. After vascular control of the upper and mid poles, ICG was administered to visualize perfusion, which clearly delineated the ischemic boundary at the planned isthmus transection site. The demarcation line was pre-marked under Firefly mode, enabling precise transection with an endoscopic stapler. The procedure was completed without blood loss, and pathology confirmed pT1 urothelial carcinoma with negative margins. To the best of our knowledge, this is the first report demonstrating the efficacy of ICG in guiding perfusion-based isthmectomy during RANU for a horseshoe kidney, and the first to provide intraoperative video documentation of contralateral renal perfusion and resection line marking. The combined use of ICG fluorescence and stapler transection enabled a safe, bloodless, and oncologically complete procedure, although further cases will be required to validate the generalizability of this technique.</p>