We aimed to introduce an approach for image-guided positioning of electrodes for irreversible electroporation (IRE) in patients with prostate cancer using a magnetic resonance imaging-transrectal ultrasonography (MRI-TRUS) fusion technique. In 10 consecutive patients with biopsy-proven Gleason score ≤3+4 prostate cancer, 19 G electrodes were inserted into the prostate using a transperineal access. Magnetic resonance images of the prostate acquired before IRE were fused with transrectal ultrasound images acquired during IRE. The position of the ultrasound probe was tracked via a sensor and corresponding magnetic resonance images were calculated in real-time. While MRI allowed delineation of the target volume, the position of the electrodes could be visualized on ultrasound images; the distance between individual electrode pairs was measured. Based on these measurements the software installed on the IRE unit was able to calculate the voltage necessary to generate the electric field for ablation. Using contrast-enhanced ultrasound, changes in perfusion within the ablation zone after IRE were documented. This technique allowed positioning of the electrodes around the target volume under image guidance in all patients treated with IRE. The target lesion and a safety margin were covered within the estimated ablation zone. MRI-TRUS guidance for IRE combines the advantages of good visualization of the target lesion on MRI with the ability of ultrasound to acquire imaging in real-time with a mobile device.