Abstract Background: Recent studies have found lower risk of breast cancer in women with higher serum concentrations of 25-hydroxyvitamin D (25[OH]D), with few exceptions. To help resolve remaining differences, a nested case-control study was conducted. Material and Methods: Serum was provided by the Department of Defense Serum Repository for 600 new cases of breast cancer and 600 controls individually matched on age ±6 months; date blood was drawn ±2 days; and active-duty status; and were analyzed for 25(OH) D by a chemiluminescent assay. Mean age was 40 years (SD ±7 years, range 20–56 years). The individuals were 55% white, 33% African-American, and 11% other races. Mean serum 25(OH)D concentrations by quintile, from lowest to highest, were 11, 18, 24, 30 and 42 ng/ml. Dividing points were <15, 15–21, 22–27, 28–35, and >35 ng/ml. Results: Race-adjusted odds ratios for breast cancer, from lowest to highest quintile of serum 25(OH)D, were 1.0 (reference), 0.58, 0.78, and 0.52 (ptrend < 0.05). The difference between risk of breast cancer in the lowest and highest 25(OH)D quintiles was significant (p < 0.05). The association of low serum 25(OH)D with high risk of breast cancer was strongest for serum drawn during the 3 month period preceding diagnosis of breast cancer, and in white women. There was a strong linear inverse relationship between serum 25(OH) D and risk of breast cancer in this group. A similar trend was present for overall data including all intervals and races, but was weaker and not as clearly linear. Discussion: Consistent with most previous research, women whose serum 25(OH)D concentration was high (42 ng/ml) had approximately half the risk of breast cancer as those whose serum 25(OH)D was low (11 ng/ml). The trend was similar after adjustment for race. The favorable association of serum 25(OH)D with risk of breast cancer apparently was strongest during the final few doublings of the tumor mass preceding diagnosis in this population. (An alternative, far less likely, interpretation, is that the tumor mass might adversely influence 25(OH)D concentration). A serum 25(OH)D concentration of 42 ng/ml can be achieved with vitamin D3 intake of 4000 IU/day. This is the safe daily upper level intake of the National Academy of Sciences-Institute of Medicine (NAS-IOM December 2010 monograph). While further research would be beneficial, such intake should be recommended as a useful tool for prevention of breast cancer. Serum 25(OH)D should be monitored on a routine basis, when feasible, to ensure that at least 40 ng/ml is maintained throughout the year, and 150 ng/ml is not exceeded. This hygienic measure could prevent approximately 50% of breast cancer in the US. (The views expressed in this abstract are those of the authors and do not represent positions of the Department of the Navy, Department of the Army, the Department of Defense or the US Government.) Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-08.