Background: Higher circulating levels of vitamin D (25(OH)D) have been associated with reduced risk of developing invasive breast cancer. However, their association with risk of ductal carcinoma in situ (DCIS) of the breast has received little attention. Methods: We examined the association of serum vitamin D with risk of DCIS in 231,203 women, aged 40-69 years at enrollment in the UK Biobank cohort study. Multivariable Cox proportional hazards models were used to estimate hazard ratios and 95% confidence intervals for the association of vitamin D with DCIS risk. Results: There was no association between serum 25(OH)D levels and risk of DCIS overall, or by menopausal status. The association was not modified by BMI category, family history of breast cancer, or current use of menopausal hormone therapy. Conclusions: In this large prospective cohort study, we did not observe an association between circulating serum levels of vitamin D and risk of DCIS. Impact: While previous studies have suggested that serum vitamin D has an inverse association with risk of invasive breast cancer, the present results do not provide evidence for an association with DCIS, a non-obligate precursor of invasive disease.