Long-term hormone therapy (HT) is a recognized risk factor for postmenopausal breast cancer. Elevated steroid hormone levels play a critical role in breast carcinogenesis and this may be contributed by the efficiency of hormone biosynthesis. Within this context, genetic polymorphisms related to steroid hormone biosynthesis may modify HT-associated postmenopausal breast cancer risk. CYP17 is a key player of this pathway and the CYP17A1_-34_T > C polymorphism has been suggested to affect breast cancer risk in women using long-term HT. We genotyped 13 polymorphisms of seven genes of the steroid hormone biosynthesis pathway in 3,149 postmenopausal breast cancer patients and 5,489 age-matched controls from Germany. We observed a significant interaction of CYP17A1_-34_T > C and HT use on breast cancer risk in a co-dominant model (P (interaction) = 0.007). Current users of estrogen monotherapy showed a significantly increased risk for duration of use per 5-year increment when they were carriers of the CYP17A1_-34_TC genotype (OR 1.13, 95% CI: 1.04-1.23 per 5 years of use). We conclude that CYP17A1_-34_T > C may be part of the genetic background to contribute to postmenopausal breast cancer risk in women using estrogen monotherapy.