Pancreatic cancer is an aggressive malignancy with high metastatic potential. There are several lifestyle-related determinants in its etiology, including diet. Methyl donors are dietary micronutrients which play an important role in fueling vital metabolic pathways, and as bioactive food components provide methyl groups as substrates and cofactors. The imbalanced nutritional status of methyl donors has recently been linked to pathological conditions. Therefore, we hypothesized that dietary methyl donors may improve the physiology of cancer patients, including those with pancreatic cancer, and could be used for intervention therapy. In this study, methyl-donor treatment (L-methionine, choline chloride, folic acid and vitamin B12) of an aggressive pancreatic adenocarcinoma cell line (Panc-1) resulted in significantly increased p21WAF1/Cip1 cyclin-dependent kinase inhibitor levels, along with apoptotic SubG1 fractions. At the same time, phospho-Erk1/2 levels and proliferation rate were significantly reduced. Though methyl-donor treatments also increased the pro-apoptotic protein Bak, Puma and Caspase-9, it failed to elevate cleaved Caspase-3 levels. In addition, the treatment significantly reduced the production of the pro-inflammatory cytokine IL-17a and the transcription factor NFkB. Similarly, a significant decrease in VEGF and SDF-1a levels were detected, which may indicate reduced metastatic potential. As expected, E-cadherin expression was inversely associated with these changes, showing elevated expression after methyl-donor treatment. In summary, we found that methyl donors may have the potential to reduce aggressive and proliferative phenotype of Panc-1 cells. This suggests a promising role of dietary methyl donors for complementing relevant cancer therapies, even in treatment-resistant pancreatic adenocarcinomas.