The objective of this study is to examine the association between vitamin D status and risk of breast cancer in an Australian population of women. The study design is observational case-control study, performed at Westmead Breast Cancer Institute, Westmead Hospital, Sydney, Australia. 214 women newly diagnosed with breast cancer were matched to 852 controls, and their blood samples were tested at the same laboratory between August 2008 and July 2010. Circulating 25-hydroxyvitamin D (25(OH)D) concentration, was defined as sufficient (≥75 nmol/L), insufficient (50-74 nmol/L), deficient (25-49 nmol/L) or severely deficient (<25 nmol/L). The difference in median 25(OH)D concentration between cases and controls was reported, and the Mann-Whitney U test was used to determine the significance of the difference. Odds ratios and 95 % confidence intervals for the risk of breast cancer were estimated by Cox regression. Median plasma 25(OH)D was significantly lower in cases versus controls overall (53.0 vs 62.0 nmol/L, P < 0.001) and during summer (53.0 vs 68.0 nmol/L, P < 0.001) and winter (54.5 vs 63.0 nmol/L, P < 0.001). Median 25(OH)D was also lower in cases when stratified by BMI (<30, ≥30) and age group (<50, ≥50 years) compared to matched controls, although the difference failed to reach statistical significance. In a Cox regression model, plasma 25(OH)D was inversely associated with the odds ratio of breast cancer. Compared to subjects with sufficient 25(OH)D concentration, the odds ratios of breast cancer were 2.3 (95 % CI 1.3-4.3), 2.5 (95 % CI 1.6-3.9) and 2.5 (95 % CI 1.6-3.8) for subjects categorised as severely deficient, deficient or insufficient vitamin D status, respectively. The results of this observational case-control study indicate that a 25(OH)D concentration below 75 nmol/L at diagnosis was associated with a significantly higher risk of breast cancer. These results support previous research which has shown that lower 25(OH)D concentrations are associated with increased risk of breast cancer.