The varying malignancy and lethality of different grades of prostate cancer (PCa) highlight the importance of accurate diagnosis. This study aims to evaluate the upgrading of transition zone (TZ) prostate cancer biopsies and identify factors to improve TZ biopsy accuracy. This retrospective study included 217 patients who underwent laparoscopic radical prostatectomy after 12 + X cores transperineal transrectal ultrasound-magnetic resonance imaging (MRI)-guided targeted prostate biopsy from 2018 to 2021 in our center. Patients with TZ lesions showed a higher incidence of International Society of Urological Pathology (ISUP) grade upgrading from 1 to higher grade compared to peripheral zone lesions (16.9% vs. 5.0%, p = 0.005). Multivariate analysis confirmed TZ lesions as an independent risk factor (OR: 4.594, 97.5% CI: 1.569-15.238, p = 0.008) for upgrading from 1 to higher. Additionally, the number of positive biopsy cores (OR: 0.586, 97.5% CI: 0.336-0.891, p = 0.029) and anterior TZ lesion location (OR: 10.797, 97.5% CI: 1.503-248.727, p = 0.048) were independent factors for the upgrading in TZ patients. This study found that PCa lesions located in the TZ, particularly the anterior TZ, have a higher risk of ISUP grade upgrading. This elevated risk arises from the insufficient distribution of biopsy cores around the TZ lesion. The findings underscore the importance of having an adequate number of biopsy cores around the lesion area to improve the accuracy of ISUP grade assessments.