Background: The use of transperineal (TP) prostate biopsy has gained in popularity due to reduced risk of infectious events, however evidence regarding cancer detection rates versus transrectal (TR) prostate biopsy is lacking. Our objective was to examine whether transperineal (TP) MRI-fusion targeted prostate biopsy (MRI-TBx) resulted in improved detection of clinically significant prostate cancer (csPCa), defined as International Society of Urological Pathology (ISUP) grade group ≥2, as compared to TR MRI-TBx. Methods: Retrospective review of all patients who underwent MRI-TBx at a tertiary care academic center. A cohort of patients who underwent TP MRI-TBx was compared to a second cohort of patients who underwent TR MRI-TBx. The primary outcome of interest was detection of csPCa, however detection of ISUP grade group 1 prostate cancer (GG1 PCa) was also examined. Analyses were performed on a lesion level basis. Multivariable logistic regression analyses were performed to assess for predictors of csPCa detection. Results: A total of 389 and 1144 lesions from 303 and 836 patients who underwent TP MRI-TBx vs TR MRI-TBx respectively were included in the analysis. The detection of both csPCa (39.6% vs 29.6%, p <0.001) and GG1 PCa (34.4% vs 21.1%, p <0.001) was higher for TP MRI-TBx vs. TR MRI-TBx. On multivariable analysis adjusted for age, prior biopsy status, Prostate Imaging–Reporting and Data System (PIRADS) score, prostate volume, PSA, size of PIRADS lesion, and zone of PIRADS lesion, TP MRI-TBx remained an independent predictor of csPCa (odds ratio [OR] 1.49, 95% CI 1.13-1.98). Surprisingly, transperineal biopsy was not a significant predictor of csPCa for Transition zone lesions (OR 1.24, p = 0.35), but was for Peripheral zone lesions (OR = 1.84, p < 0.001). Conclusions: Performance of TP MRI-TBx demonstrates improved detection of both csPCa and GG1 PCa. This difference is not dependent on anterior/transition zone location. This has important implications in risk assessment for localized prostate cancer, including counseling for active surveillance and definitive management strategies.