The study aimed to evaluate the learning curve associated with transperineal magnetic resonance imaging (MRI) and transrectal ultrasound (TRUS) fusion-guided prostate biopsy (PBx). A total of 370 consecutive men who underwent MRI followed by transperineal PBx were prospectively enrolled and categorized into five chronological quintiles, with each group comprising 74 patients. All participants underwent a systematic biopsy, with additional targeted biopsies taken for those with PIRADS scores of 3 or higher. The procedure initially used sedation in the first quintile and transitioned to local anesthesia for the subsequent groups. The researchers utilized inflection point analysis to identify when proficiency in operative time was achieved, considering clinically significant prostate cancer (CSPCa) as Grade Group 2 or higher. The detection rates of CSPCa across the quintiles showed no significant variation, indicating the consistent effectiveness of the biopsy method. However, operative times decreased markedly from an average of 45 minutes in the first quintile to 19 minutes by the fifth quintile, reaching a plateau after 147 cases. The pain experienced by patients immediately after the procedure did not significantly differ across the later quintiles, and the complication rates remained low and consistent throughout the study. Overall, while CSPCa detection rates were adequate from the outset, significant improvements in operative time and consistency in patient experience highlight the learning curve associated with this innovative biopsy technique.