We prospectively investigated the clinical parameters that influenced pain during prostate biopsies. From 12 hospitals 1781 patients were enrolled. The patients completed a visual analogue scale questionnaire for the pain during the procedure. Age, enema preparation, analgesia use and number of biopsy punctures influenced the level of pain during prostate biopsy in univariate linear regression analysis. However, multivariate analysis showed enema preparation, analgesia use and number of biopsy punctures were independent factors associated with the pain during the procedure. Our study confirmed enema preparation before biopsy and the number of biopsy punctures were associated with the pain during prostate biopsy.