Several laboratory and epidemiological studies have inversely linked endogenous vitamin D and the risk of breast cancer. The acquisition of vitamin D over time on the relative risk (RR) of the disease development is not known. In a longitudinal study, we evaluated the association between vitamin D levels at pregnancy over time with the risk of breast cancer, and pregnancy-associated breast cancer.The risk for subsequent development of breast cancer associated with serum 25-hydroxyvitamin (25-OHD) levels was assessed for consecutive (1st and 2nd pregnancy) samples of 100 cases, with mean lag times (μ t ) of 7.4 and 4.6 years between sampling and the diagnosis, and matched (parity, age, year, season) controls. Pregnancy-associated breast cancer (PABC, 111 case–control pairs, μ t =1year) risk was also studied. Odds ratios (ORs) with 95% confidence intervals (CI) were calculated using the lowest quintile as the reference.Serum 25-OHD level was not associated with an increased risk neither at the 1st nor at the 2nd pregnancy samples (OR=1.4, 95%CI 0.6–3.4; OR 1.4, 95%CI 0.7–2.8, respectively), but was associated with an increased risk of PABC (OR=2.7, 95%CI 1.04–6.7).Generally, vitamin D may not be related to breast cancer risk but the increased PABC risk fits the association of vitamin D with the most aggressive cancers, and warrants caution with vitamin D supplementation during pregnancy.