MRI-guided prostate biopsy in conventional closed-bore scanners requires transferring the patient outside the bore during needle insertion due to the constrained in-bore space, causing a safety hazard and limiting image feedback. To address this issue, we present our custom-made in-bore setup and software to support MRI-guided transperineal prostate biopsy in a wide-bore 3 T MRI scanner. The setup consists of a specially designed tabletop and a needle-guiding template with a Z-frame that gives a physician access to the perineum of the patient at the imaging position and allows the physician to perform MRI-guided transperineal biopsy without moving the patient out of the scanner. The software and Z-frame allow registration of the template, target planning and biopsy guidance. Initially, we performed phantom experiments to assess the accuracy of template registration and needle placement in a controlled environment. Subsequently, we embarked on our clinical trial (N = 10). The phantom experiments showed that the translational errors of the template registration along the right-left (RP) and anterior-posterior (AP) axes were 1.1 ± 0.8 and 1.4 ± 1.1 mm, respectively, while the rotational errors around the RL, AP and superior-inferior axes were (0.8 ± 1.0)°, (1.7 ± 1.6)° and (0.0 ± 0.0)°, respectively. The 2D root-mean-square (RMS) needle-placement error was 3 mm. The clinical biopsy procedures were safely carried out in all ten clinical cases with a needle-placement error of 5.4 mm (2D RMS). In conclusion, transperineal prostate biopsy in a wide-bore 3T scanner is feasible using our custom-made tabletop setup and software, which supports manual needle placement without moving the patient out of the magnet.