Aims and Background The importance of optimal daily patient positioning has been stressed in order to ensure treatment reproducibility and gain in accuracy and precision. We report our data on the 3D setup uncertainty during radiation therapy for prostate cancer using the CT image fusion technique. Methods Ten consecutive patients scheduled for radiation therapy for prostate cancer underwent 5 prone position CT scans using an individualized immobilization cast. These different setups were analyzed using the image fusion module of the ERGO 3D-Line Medical System (Milan, Italy) treatment planning system. The isocenter and the body marker displacements were measured. Results The 3D isocenter dislocations were quantified: systematic error was Σ3D = 3.9 mm, whereas random error was σ3D = 1 mm. The mean of the minimum displacements was 0.2 ± 1 mm showing that the immobilization device used allows an accurate setup to be obtained. Single direction errors were also measured showing systematic errors, ΣAP = 2.6 mm, ΣLL = 0.6 mm, ΣSI = 3 mm in the anterior-posterior, latero-lateral, superior-inferior direction, respectively. Related random errors were σAP = 1 mm, σLL = 0.6 mm, σSI = 1.2 mm. In terms of accuracy, our uncertainties are similar to those reported in the literature. Conclusions By applying the CT image fusion technique, a 3D study on setup accuracy was performed. We demonstrated that the use of an individualized immobilization system for prostate treatment is adequate to obtain good setup accuracy, as long as a high-quality positioning control method, such as the stereoscopic X-ray-based positioning system, is used.