Introduction: We correlated serum 25-hydroxyvitamin D3 (25OHD) levels with tumor characteristics and clinical disease outcome in breast cancer patients and assessed the impact of genetic determinants of vitamin D insufficiency.Methods: We collected serum from 1800 early breast cancer patients at diagnosis, measured 25OHD by radioimmunoassay and determined genetic variants in vitamin D-related genes by Sequenom. Multivariable regression models were used to correlate 25OHD levels with tumor characteristics. Cox proportional hazard models were used to assess overall survival (OS), disease-specific survival (DSS) and disease-free interval (DFI).Results: Lower 25OHD serum levels significantly correlated with larger tumor size at diagnosis (p=0.0063) but not with lymph node invasion, receptor status, or tumor grade. Genetic variants in 25-hydroxylase (CYP2R1) and vitamin D-binding protein significantly determined serum 25OHD levels but did not affect the observed association between serum 25OHD and tumor size. High serum 25OHD (>30 ng/mL) at diagnosis significantly correlated with improved OS (p=0.0101) and DSS (p=0.0192) and additionally had a modest effect on DFI, which only became apparent after at least 3 years of follow-up. When considering menopausal status, serum 25OHD had a strong impact on breast cancer-specific outcome in postmenopausal patients (hazards ratios for 25OHD >30 ng/mL versus ≤30 ng/mL were 0.15 [p=0.0097] and 0.43 [p=0.0172] for DSS and DFI, respectively), whereas no association could be demonstrated in premenopausal patients.Conclusion: High vitamin D levels at early breast cancer diagnosis correlate with lower tumor size and better OS, and improve breast cancer-specific outcome, especially in postmenopausal patients.