Background: Appropriate risk stratification of men on active surveillance for prostate cancer is essential to identify men in whom it is safe to take this deferred treatment approach. This study evaluates upstaging rates using MRI-US fusion targeted biopsy in men who have had a prior positive standard 12-core biopsy. Methods: Between 12/2012 and 06/2015, 374 men with an indication for prostate biopsy underwent pre-biopsy mpMRI followed by 12-core standard trans-rectal mapping biopsy (Mbx) and MRI-Ultrasound fusion targeted biopsy (Tbx) of lesions identified on mpMRI. The combination of Mbx and Tbx, when both occurred, constitutes a fusion biopsy (Fbx). Men who underwent both Mbx with or without Tbx using the Artemis/Pro-Fuse system with a previous non-MRI-guided biopsy and a diagnosis of prior Gleason 6 prostate cancer were included. Patients without a lesion on MRI underwent Mbx only. Maximum Gleason scores (GS) were assigned on a per patient basis with Mbx GS available for all patients in the cohort and Tbx GS available only for patients with a lesion visible on MP-MRI. Clinically significant (CS) cancer was defined as GS ≥ 3+4. GS per patient was compared by chi-square and McNemar’s test. Results: 118 patients met inclusion criteria (Mean PSA = 6.9, Mean age = 62.5). 40 patients (34%) were upstaged by Fbx to Gleason ≥ 7. Of those upstaged, 17 men (14%) would have been missed by Mbx alone, in comparison to 7 (6%) that were missed by Tbx alone. Total number of prior biopsies (p = 0.28) and number of years on Active Surveillance (p = 0.22) were not related to upgrade on Fbx. Older men (65.3 vs. 60.9, p = 0.033) and those with higher PSA (8.7 vs 5.8, p = 0.002) were more likely to be upgraded on Fbx. Tbx was more likely to identify CS cancer than Mbx (85% vs 56%; p < 0.012). Conclusions: MP-MRI Fusion biopsy more accurately stratifies men with a previous prostate biopsy than those receiving a template mapping 12-core biopsy alone. Tbx should be strongly considered before enrolling a patient in active surveillance since up 14% of clinically significant cancer would have been missed with a 12-core biopsy alone.