Background: The common HSD3B1 (1245A > C) germline variant is associated with increased de-novo synthesis of androgens and worse outcomes in men treated with androgen-deprivation therapy in metastatic hormone sensitive prostate cancer. The aim of this study is to determine the role of this polymorphism on treatment outcomes for AA and ENZA in patients with mCRPC. Methods: A total of 547 patients treated with AA or ENZA for mCRPC from two prospective cohorts; cohort 1 included 202 from British Columbia (Canada) and cohort 2 enrolled 345 patients from the Spanish study PROREPAIR-B. HSD3B1 genotype was determined by targeted sequencing in cohort 1 and by Taqman SNP genotyping assay in cohort 2. Associations between HSD3B1 genotypes and (TTPP), time to progression (TTP) and overall survival (OS) were evaluated via univariate COX regression. Multivariate analysis was performed to determine the independent association of each covariate. Results: The proportions of patients with a homozygous wild-type HSD3B1 (AA), heterozygous (AC) and homozygous variant (CC) genotype were respectively 45.6%, 39.4% and 15%. As expected, known prognostic factors for mCRPC such as hemoglobin, alkaline phosphatase (ALP), LDH, PSA at baseline as well as site of metastasis were significantly associated with TTPP and TPP. In the combined cohort, HSD3B1 (CC) genotype was associated with worse TTP (HR 1.31, 95%CI 1.02-1.67, p = 0.032) and PSA response rates (48% for CC vs 62% and 65% for AA and AC, respectively (p = 0.019, χ²)). Similar trend was observed for TTPP (HR 1.28, 95%CI 0.99-1.66, p = 0.064). OS was not different among genotypes, but was significantly shorter for patients with CC genotype in cohort 1 (HR 1.97, 95%CI 1.14-3.40, p = 0.016). There was no association between HSD3B1 genotype and time to castration-resistance in either of the two cohorts. Multivariable analysis showed that LDH, ALP, hemoglobin and use of AA or ENZA as first-line therapy for mCRPC were independent prognostic factors for TTP and TTPP; non-significant association was observed for genotype and TTP. Conclusions: HSD3B1 homozygous variant genotype (CC) was associated with shorter TTP and lower PSA response rate in mCRPC patients treated with AA or ENZA. However, the CC genotype did not provide prognostic information beyond that conferred by standard clinical variables, suggesting that it may not be a suitable stand-alone biomarker in mCRPC.