Background: The enzyme encoded by the HSD3B1(1245C) variant allele has been shown to promote castration-resistant prostate cancer by increasing intratumoral dihydrotestosterone synthesis. In the setting of biochemical recurrence (BCR) following prostatectomy, inheritance of the variant allele has been associated with inferior clinical outcomes for men treated with androgen deprivation therapy (ADT). Whether the same is true in the context of BCR after radiotherapy (RT) is unknown. Methods: We determined HSD3B1 genotype retrospectively in men treated with ADT for post-RT BCR using an established biorepository at a large academic center. We analyzed time-to-progression (TTP), time-to-metastasis (TTM), and overall survival (OS) according to HSD3B1genotype using an additive genetic model with the log-rank trend test. Multivariable analyses (MVA) were performed to adjust for known prognostic factors with Cox regression. Results: We identified 218 men treated with ADT for BCR after RT, of whom 213 (98%) were successfully genotyped (46%, 45% and 9% carrying 0, 1, and 2 variant alleles, respectively). Median follow-up was 7.9 years (yrs). Demographic and treatment factors were similar across genotypes. Median TTP was 2.3 (95% CI: 1.6, 3.1) yrs in men who inherited 0 variant alleles, 2.3 (1.5, 3.3) yrs with 1 variant allele, and 1.4 (0.7, 3.3) yrs with 2 variant alleles (P = 0.683). Median TTM diminished with the number of variant alleles inherited (7.4 [6.7, 9.7], 5.8 [4.9, 6.5] and 4.4 [3.0, 5.7] yrs, respectively (P = 0.030). No difference in OS was detected (P = 0.305). On MVA with 0 variant alleles as the reference, the adjusted hazard ratio (HR) for metastasis was (1.19 [0.74, 1.92]; P = 0.480) for 1 allele and (2.01 [1.02, 3.97]; P = 0.045) for 2 alleles. MVA did not demonstrate significant differences in TTP or OS. Conclusions: The HSD3B1(1245C) allele that enhances dihydrotestosterone synthesis is associated with time-to-metastasis in men treated with ADT for BCR after RT for prostate cancer. Notably, 49% of men had received prior ADT as part of local therapy and 56% received an anti-androgen during ADT for BCR, which may blunt the effect of the variant allele.