ABSTRACT Introduction Considering the distinctive nature in terms of psychological stress and anal tone of position which is generally selected between lithotomy and left lateral decubitus (LLD), we postulated its effect on pain perception during biopsy, and investigated their association. Materials and Methods A prospective study for comparison of two biopsy positions which were perform in a different working day was conducted for 208 men (lithotomy position=86, LLD=122). The decision on the position was made solely based on the patient’s preference for the biopsy day, and all procedures were performed according to the identical protocol (12-core biopsy with intrarectal lidocaine gel), probe, and needle. The maximal degree of pain during the entire process was assessed using a visual analogue scale (VAS), immediately after biopsy. After propensity matching, a total of 152 patients were finally selected (lithotomy group=76, LLD=76), then peri-biopsy parameters were compared. Results Between groups, no differences were observed across all variables including age, obesity, prostate volume, serum PSA, international prostate symptom score, and cancer detection rate, except mean (±standard deviation) VAS score (3.89±2.01 vs. 4.58±2.22, p=0.049). VAS score showed significant association solely with patient’s position (Pearson’s coefficient=-0.165, p=0.042). In multiple linear regression models regarding the effect of clinical variables on VAS score, patient position was a single independent predictor favoring lithotomy position to decrease perceived pain (B=-0.928, p=0.024). Conclusions These data suggest lithotomy position as a proper way to perform transrectal prostate biopsy with routine use of topical lidocaine gel in comparison with conventional LLD position.