Abstract Background Epigenome-wide association studies (EWAS) using peripheral blood have identified specific sites of DNA methylation associated with risk of various cancers and may hold promise to identify novel biomarkers of risk; however, few studies have been performed for pancreatic cancer and none using a prospective study design. Methods Using a nested case-control study design, incident pancreatic cancer cases and matched controls were identified from participants who provided blood at baseline in three prospective cohort studies. DNA methylation levels were measured in DNA extracted from leukocytes using the Illumina MethylationEPIC array. Average follow-up period for this analysis was 13 years. Results Several new genomic regions were identified as being differentially methylated in cases and controls; the five strongest associations were observed for CpGs located in genes TMEM204/IFT140, MFSD6L, FAM134B/RETREG1, KCNQ1D and C6orf227. For some CpGs located in chromosome 16p13.3 (near genes TMEM204 and IFT140), associations were stronger with shorter time to diagnosis (e.g., OR = 5.95, 95% CI = 1.52-23.12, for top vs bottom quartile, for <5 years between blood draw and cancer diagnosis) but associations remained statistically significantly higher even when cases were diagnosed over 10 years after blood collection. Statistically significant differences in DNA methylation levels were also observed in the gastric secretion pathway using GSEA analysis. Conclusions Changes in DNA methylation in peripheral blood may mark alterations in metabolic or immune pathways that play a role in pancreatic cancer. Identifying new biological pathways in carcinogenesis of pancreatic cancer using EWAS approach could provide new opportunities for improving treatment and prevention.