17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) plays an important role in the biosynthesis of 17β-estradiol. The current study aimed at confirming the reduced risk of breast cancer in carriers of the non-synonymous HSD17B1_937_A>G (rs605059) polymorphism who used any hormone replacement therapy (HRT) for 10 years or longer. We performed an independent association study using four breast cancer case-control studies from Australia, Germany, and Sweden. In all, 5,777 cases and 8,189 age-matched controls of European descent were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and TaqMan. Risk estimates were calculated by interaction analysis and main effect analysis adjusted for age and study. Main effect analyses for women using any HRT for 10 years or longer (1,428 cases versus 1,724 controls) revealed a protective effect of the HSD17B1_937_G allele on breast cancer risk (OR 0.86, 95 % CI: 0.73-0.99; p = 0.048). Thus, our previous finding of a protective effect of the HSD17B1_937_G allele on HRT-associated breast cancer risk has now been confirmed both in independent large patient cohorts and a comprehensive pooled analysis supporting the hypothesis that a HSD17B1-mediated decreased conversion of estrone to the more potent 17β-estradiol may reduce the estrogenic effects, thereby reducing the risk of developing breast cancer during long-term HRT use.