Purpose Horseshoe kidneys pose operative challenges due to atypical renal anatomy and vasculature. This study demonstrates how three-dimensional (3D) virtual reconstruction comprehensively represents renal and tumour anatomy to inform operative approach in robotic-assisted partial nephrectomy (RAPN) in horseshoe kidneys. Methodology Case selection included patients at Nepean Hospital with horseshoe kidney tumours amenable to RAPN. 3D virtual reconstruction of the kidney was generated from CT images and consulted pre-operatively. RAPN was performed with the da Vinci® Surgical System. Results A 74-year-old male with a 35 mm x 26 mm partially endophytic complex left upper pole horseshoe kidney cyst and no evidence of metastatic disease was selected for this study. The 3D virtual reconstruction revealed renal vasculature of two left renal arteries and one left renal vein dividing proximally, which informed the dissection approach and decision to clamp all three vessels prior to tumour excision. The 3D reconstruction revealed tumour proximity to the pelvicalyceal system allowing preparation for a collecting system defect following excision. The 3D reconstruction outlined the vascular supply of the isthmus and right side of the kidney as arising from the aorta inferior to the inferior mesenteric artery to facilitate safety planning in the event of haemorrhage. The tumour was classified as grade 2 clear cell renal cell carcinoma of stage pT1a. Clear surgical margins were achieved. Conclusion The 3D virtual reconstruction provided a more comprehensive visual representation of horseshoe kidney anatomy than CT. 3D virtual reconstruction facilitates precise tumour excision and minimises devascularisation of the renal parenchyma.