Background: The utility of serial magnetic resonance imaging (MRI)/ultrasound (US) fusion targeted prostate biopsy in men with prostate cancer (PCa) on active surveillance (AS) have not been clearly defined. We sought to investigate the rate of Gleason upgrading both on sequential fusion targeted and systematic biopsies among men with low-risk PCa managed with AS. Methods: We retrospectively queried an institutional database of 800 patients undergoing MRI/US fusion biopsy to identify 209 patients on AS with at least two fusion biopsies between December 2013 and November 2016. Men with National Comprehensive Cancer Network (NCCN) very low-risk and low-risk criteria were included. Gleason upgrade was defined as detection of Gleason score >=3+4. The proportion of patients experiencing upgrade on systematic, fusion, or both biopsy techniques was tabulated. Associations of clinical, pathologic, and imaging factors with biopsy upgrade were analyzed by logistic regression. Results: Of 209 patients undergoing MRI/US fusion biopsy, 73 (35.0%) had at least two targeted biopsies (66% very low-risk and 34% low-risk PCa). The time between biopsies was 12.6 months (11.2-17.7). The median PSA and PSA density were 5.4 ng/mL (4.2-7.1) and 0.11 ng/mL/mL (0.07-0.18), respectively. 21 (29%) patients experienced Gleason upgrade on subsequent biopsy. Of those, 6 (8%), 5 (7%), and 10 (14%) had upgrade on systematic biopsy only, fusion biopsy only, and both systematic and fusion biopsy, respectively. Patients with upgrade on subsequent biopsy had higher PSA (p=0.02) and PSA density (p=0.02), and were among low-risk disease population (p=0.03). In logistic regression models, greater number of positive cores in systematic biopsy (OR 1.88; 95% CI 1.23-2.92; p=0.005) was associated with the total Gleason upgrade on repeated biopsy. Conclusions: In men with favorable risk prostate cancer managed with AS, Gleason upgrade was detected in a 29% of patients on a second MRI/US fusion biopsy including both targeted and systematic regions. These findings support the continued use of both MRI fusion and systematic biopsy during surveillance due to risks of reclassification over time.