Endogenous hormones play a role in many health conditions that impact women. Estrogens increase breast cancer risk through estrogen receptor (ER) mediated pathway activation. Modifiable factors such as 25-hydroxyvitmain D (25(OH)D) may influence endogenous hormone levels, and incidence and survival of diseases such as breast cancer. In Chapter 1, we conducted a prospective, nested case-control study among postmenopausal women using the Nurses’ Health Study (NHS), that included 371 cases with blood samples collected prior to breast cancer diagnosis and 731 matched controls. Estrogen pathway activity (EA) was assessed via a luciferase reporter assay using T47D-Kbluc human breast cancer cells. We assessed the contribution of EA to risk, independent of circulating estrone, estradiol, and estrone sulfate concentrations. Our study provides a first detailed assessment of a breast cancer cell line-based EA assay and postmenopausal breast cancer risk. In Chapter 2, we examined the association between circulating 25(OH)D concentrations and breast cancer-specific and all-cause mortality among women with breast cancer. Our study population consisted of 1,312 invasive breast cancer survivors in the NHS and NHS2 cohorts. Women provided a blood sample in 1989-1990 and a second blood sample in 2000-2001 (NHS) or a blood sample in 1996-1999 (NHS2). Women were diagnosed with breast cancer after blood collection and followed until death or 2016 (NHS) or 2017 (NHS2). In Chapter 3, we evaluated the association of 25(OH)D and vitamin D binding protein (DBP) with levels of several endogenous sex steroids and prolactin to provide insight into the potential biologic impact of 25(OH)D and DBP. We conducted a cross-sectional study among 1,625 premenopausal NHS2 women. We used 25(OH)D and DBP concentrations and menstrual cycle-timed sex hormones, all measured in plasma. Estrogen metabolite levels were measured in luteal phase urine samples. In conclusion, our findings suggested (1) a positive association between EA and breast cancer risk, however, the association was substantially attenuated after accounting for levels of other estrogens, (2) higher pre-diagnostic vitamin D levels are associated with improved overall survival, but not breast cancer-specific survival, in women with breast cancer, and (3) little association of 25(OH)D or DBP with sex steroid hormones.