Background: Folate plays a critical role in DNA methylation, synthesis, and repair. Several epidemiologic studies suggest that higher folate intake is associated with decreased pancreatic cancer risk. Objective: We investigated the association between dietary folate intake and pancreatic cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) cohort. Design: Dietary data were collected with the use of a self-administered food-frequency questionnaire (1998-2005). Among the 51,988 male and 57,187 female participants, aged 55-74 y at enrollment, with complete dietary and multivitamin information, 162 men and 104 women developed pancreatic cancer during follow-up (January 1998 to December 2006; median: 6.5 y). We used Cox proportional hazards regression with age as the time metric to calculate hazard ratios (HRs) and 95% CIs. Results: The highest compared with the lowest quartile of food folate was associated with a significantly decreased pancreatic cancer risk among women (≥253.3 compared with ≤179.1 μg/d; HR = 0.47; 95% CI: 0.23, 0.94; P for trend: 0.09) but not among men (≥229.6 compared with ≤158.0 μg/d; HR = 1.20; 95% CI: 0.70, 2.04; P for trend: 0.67; P for interaction by sex: 0.03). There was also a significant inverse trend in risk of pancreatic cancer across increasing quartiles of total folate in women (P for trend: 0.04) but not in men (P for trend: 0.65). Folic acid supplements were not associated with pancreatic cancer. Conclusion: These findings support an association between higher food and total folate intakes and decreased risk of pancreatic cancer in women but not in men.