Background: Exposure to Agent Orange (AO), a defoliate used during the Vietnam War, is a potential risk factor for the development of prostate cancer (PCa). However, it is unknown whether individuals exposed to AO are at risk of a more aggressive phenotype of PCa than individuals not exposed to AO. The goal of this study was to determine the association between AO exposure and risk of high-grade PCa in a cohort of veterans referred for an initial prostate biopsy. Methods: Risk factors were identified using historical clinical data from individuals referred to the Portland VA Hospital for a prostate biopsy between 1993 and 2010. Covariates evaluated include AO exposure, prostate-specific antigen density (PSAD), digital rectal exam (DRE), age, family history, body mass index (BMI), race, and service history. Outcomes of the biopsies were defined as high-grade PCa (HGPCa, Gleason ≥ 7) vs. other [low-grade PCa (LGPCa, Gleason ≤ 6) or no PCa]. Multivariate logistic regression was used to estimate the risk of HGPCA in those with AO exposure. To determine whether AO exposure increased the risk of both HGPCa and LGPCa, separate multivariate models compared the odds of AO exposure in individuals with HGPCa or LGPCa vs. no PCa. Results: Of the 2720 veterans who underwent prostate biopsy, 896 (32.9%) were found to have PCa and 459 (16.9%) were found to have HGPCa. After adjustment for significant confounders including PSAD, DRE, and age, HGPCa cases were more likely to have been exposed to AO as compared to those with LGPCa or no PCa (aOR = 1.73, 95% CI: 1.14 to 2.62). In an analysis of HGPCa vs. no PCa, AO was strongly associated with HGPCa (aOR = 1.74, 95% CI: 1.12 to 2.73). In contrast, in an analysis of LGPCa vs. no cancer, AO exposure was not associated with the detection of LGPCa (aOR = 1.21, 95% CI: 0.80 to 1.85). Thus, AO exposure appeared to uniquely increase the risk of HGPCa in men presenting for an initial prostate biopsy. Conclusions: Agent Orange exposure was associated with a significant increase in the risk of HGPCa in men referred for an initial prostate biopsy. If validated, these findings could aid in the development of effective PCa screening strategies for Vietnam-era veterans.