High concentrations of circulating 25-hydroxyvitamin D [25(OH)D] have been associated with elevated pancreatic cancer risk. As this is contrary to an expected inverse association between vitamin D status and cancer, we examined whether vitamin D binding protein (DBP), the primary carrier of vitamin D compounds in circulation, plays a role in this relationship. Prediagnostic serum DBP and 25(OH)D were studied in relation to risk of pancreatic cancer in a nested case-control study of 234 cases and 234 controls in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study of Finnish men. ORs and 95% CIs were estimated using logistic regression, and statistical tests were two-sided. We found that DBP and 25(OH)D were correlated (r = 0.27, P < 0.0001), and DBP was inversely associated with pancreatic cancer risk (OR = 0.66, 95% CI = 0.39-1.12, for the highest vs. lowest quartile; P(trend) = 0.02). Importantly, this association seemed to have a threshold between quartiles 2 to 4 and quartile 1, and was primarily evident among men with concurrent high 25(OH)D concentrations (OR = 0.33, 95% CI = 0.16-0.70 for highest vs. lowest quartile; P(trend) = 0.002), with no association in men with lower serum 25(OH)D (OR = 1.28, 95% CI = 0.62-2.61 for highest vs. lowest quartile, P(trend) 0.63, P(interaction) = 0.01). Men with higher 25(OH)D concentrations and serum DBP below the median showed greatly elevated risk of pancreatic cancer (OR = 5.01, 95% CI 2.33-10.78, for highest vs. lowest quartile; P(trend) < 0.0001), while risk was weakly inversely associated with serum 25(OH)D when DBP concentrations were higher (P(interaction) = 0.001). Taken together, our findings indicate that higher DBP concentrations may sequester more 25(OH)D and reduce free 25(OH)D bioavailability. Simultaneous examination of DBP and 25(OH)D may be important in determining the association of vitamin D with cancer risk.