Background: The 3β-HSD1 protein, encoded by the HSD3B1 gene, catalyzes the rate-limiting step necessary of non-testicular testosterone and dihydrotestosterone production. The common adrenal-permissive HSD3B1(1245C) allele is responsible for encoding 3β-HSD1 protein with decreased susceptibility to degradation resulting in increased of extragonadal androgen synthesis. Retrospective studies have suggested an association with the HSD3B1 CC genotype and resistance in prostate cancer. Methods: We conducted a cohort analysis of patients with a diagnosis of prostate cancer enrolled in the Million Veteran Program with genotyping and phenotyping information available through Veterans Health Administration. The primary endpoint was to evaluate prostate cancer-specific mortality (PCSM) in the overall cohort. PCSM was defined as the time from diagnosis to death from prostate cancer, censored at the date of last VA follow-up. Secondary endpoints included incidence of metastases and PCSM in predefined subgroups. HSD3B1 genotype status was categorized as AA (wild type), AC (heterozygous), CC (homozygous). Results: The study included 5287 men with prostate cancer; 402 (7.6%) HSD3B1 CC, 1970 (37.3%) HSD3B1 AC, and 2915 (55.1%) HSD3B1 AA. The cohort included 29.6% (n=1567) Black men of whom 1.2% (n=19) were HSD3B1 CC. Cumulative incidence of PCSM at five years after prostate cancer diagnosis was higher among men homozygote for HSD3B1 variant genotype CC compared to heterozygotes AC and wild-type homozygotes AA (1.9% for AA vs. 2.1% for AC vs. 4.0% for CC, p<0.017). While the cumulative incidence of metastases was similar in between HSD3B1 genotype groups (12.3% for AA, 11.8% for AC, 13.2% for CC, p=0.789), in patients who developed metastatic disease (n=619), the cumulative incidence of PCSM at five years was higher in the HSD3B1 CC group compared to AC and CC (19.2% for AA vs. 17.6% for AC vs. 37.3% for CC, p<0.005). Conclusions: Among men with prostate cancer, HSD3B1 CC genotype is associated with inferior outcomes. The HSD3B1 biomarker may help identify patients who may benefit from therapeutic targeting of 3β-HSD1 and the androgen-signaling axis.