Abstract Background: Although vitamin D inhibits breast tumor growth in experimental settings, the findings from population-based studies remain inconclusive. Our goals were to investigate the association between prediagnostic plasma 25-hydroxyvitamin D [25(OH)D] concentration and breast cancer recurrence in prospective epidemiologic studies and to explore the molecular underpinnings linking 25(OH)D to slower progression of breast cancer in the Nurses' Health Studies (NHS, N = 659). Methods: Plasma 25(OH)D was measured with a high-affinity protein-binding assay and a radioimmunoassay. We profiled transcriptome-wide gene expression in breast tumors using microarrays. Hazard ratios (HR) of breast cancer recurrence were estimated from covariate-adjusted Cox regressions. We examined differential gene expression in association with 25(OH)D and employed pathway analysis. We derived a gene expression score for 25(OH)D, and assessed associations between the score and cancer recurrence. Results: Although 25(OH)D was not associated with breast cancer recurrence overall [HR = 0.97; 95% confidence interval (CI), 0.88–1.08], the association varied by estrogen-receptor (ER) status (Pinteraction = 0.005). Importantly, among ER-positive stage I to III cancers, every 5 ng/mL increase in 25(OH)D was associated with a 13% lower risk of recurrence (HR = 0.87; 95% CI, 0.76–0.99). A null association was observed for ER-negative cancers (HR = 1.07; 95% CI, 0.91–1.27). Pathway analysis identified multiple gene sets that were significantly (FDR < 5%) downregulated in ER-positive tumors of women with high 25(OH)D (≥30 ng/mL), compared with those with low levels (<30 ng/mL). These gene sets are primarily involved in tumor proliferation, migration, and inflammation. 25(OH)D score derived from these gene sets was marginally associated with reduced risk of recurrence in ER-positive diseases (HR = 0.77; 95% CI, 0.59–1.01) in the NHS studies; however no association was noted in METABRIC, suggesting that further refinement is need to improve the generalizability of the score. Conclusions: Our findings support an intriguing line of research for studies to better understand the mechanisms underlying the role of vitamin D in breast tumor progression, particularly for the ER-positive subtype. Impact: Vitamin D may present a personal-level secondary-prevention strategy for ER-positive breast cancer.