Vitamin D may protect against several cancers, but data about the association between circulating vitamin D and bladder cancer are limited. Within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, a randomized controlled trial conducted to determine the effects of alpha-tocopherol and beta-carotene supplements on cancer incidence in male smokers, 250 bladder cancer cases were randomly sampled by month of blood collection. Controls were matched 1: 1 to cases on age at randomization and date of blood collection. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) of bladder cancer by a priori categories of baseline serum 25-hydroxyvitamin D [25(OH) D; i.e., <25, 25 to <37.5, 37.5 to <50, >= 50 nmol/L] and by season-specific quartiles. After multivariable adjustment, we found that lower 25(OH) D was associated with a statistically significantly increased risk of bladder cancer (versus >= 50 nmol/L; <25 nmol/L: OR, 1.73; 95% CI, 1.03-2.91; 25 to <37.5 nmol/L: OR, 1.81; 95% CI, 1.05-3.14; 37.5 to <50 nmol/L: OR, 1.76; 95% CI, 1.02-3.02; P trend = 0.04). Similarly, increased risks for the lowest vitamin D category were observed when season-specific quartiles were used (Q1 versus Q4: OR, 1.63; 95% CI, 0.96-2.75; P trend = 0.03). In this prospective study of male smokers, lower serum 25(OH) D was associated with an increased risk of bladder cancer. Future studies should examine the association in other populations, especially nonsmokers and women. Cancer Res; 70(22); 9218-23. (C) 2010 AACR.