Laboratory studies suggest that vitamin D (vitD) enhances chemotherapy- induced cell death. The objective of this study was to determine whether pretreatment vitD levels were associated with response to neoadjuvant chemo- therapy (NACT) in women with breast cancer. Study patients (n = 82) were enrolled on the I-SPY TRIAL, had HER2-negative tumors, and available pre- treatment serum. VitD levels were measured via DiaSorin radioimmunoassay. The primary outcome was pathologic residual cancer burden (RCB; dichoto- mized 0/1 vs. 2/3). Secondary outcomes included biomarkers of proliferation, differentiation, and apoptosis (Ki67, grade, Bcl2, respectively) and 3-year relapse-free survival (RFS). Mean and median vitD values were 22.7 ng/mL (SD 11.9) and 23.1 ng/mL, respectively; 72% of patients had levels deemed “insufficient” (<30 ng/mL) by the Institute of Medicine (IOM). VitD level was not associated with attaining RCB 0/1 after NACT (univariate odds ratio [OR], 1.01; 95% CI, 0.96–1.05) even after adjustment for hormone receptor status (HR), grade, Ki67, or body mass index (BMI). Lower vitD levels were associ- ated with higher tumor Ki67 adjusting for race (OR, 0.95; 95% CI, 0.90–0.99). VitD level was not associated with 3-year RFS, either alone (hazard ratio [HzR], 0.98; 95% CI, 0.95–1.02) or after adjustment for HR, grade, Ki-67, BMI, or response. VitD insufficiency was common at the time of breast cancer diag- nosis among women who were candidates for NACT and was associated with a more proliferative phenotype. However, vitD levels had no impact on tumor response to NACT or short-term prognosis.