Consumption of polluted fish may lead to high levels of persistent organic pollutants (POPs) in humans, potentially causing adverse health effects. Altered DNA methylation has been suggested as a possible contributor to a variety of adverse health effects. The aim of this study was to evaluate the relationship between serum POP levels (dioxins, polychlorobiphenyls, and perfluoroctane sulphonate) and DNA methylation. We recruited a total of 80 Dutch men who regularly consumed eel from either low- or high-polluted areas, and subsequently had normal or elevated POP levels. Clinical parameters related to e.g. hormone levels and liver enzymes were measured as biomarkers for adverse health effects. The Infinium 450K BeadChip was used to assess DNA methylation in a representative subset of 34 men. We identified multiple genes with differentially methylated regions (DMRs; false discovery rate <0.05) related to POP levels. Several of these genes are involved in carcinogenesis (e.g. BRCA1, MAGEE2, HOXA5), the immune system (e.g. RNF39, HLA-DQB1), retinol homeostasis (DHRS4L2), or in metabolism (CYP1A1). The DMRs in these genes show mean methylation differences up to 7.4% when comparing low- and high-exposed men, with a mean difference up to 14.4% for single positions within a DMR. Clinical parameters were not significantly associated with serum POP levels. This is the first explorative study investigating extensive DNA methylation in relation to serum POP levels among men. We observed that elevated POP levels are associated with aberrant DNA methylation profiles in adult men who consumed high-polluted eel. These preliminary findings warrant further confirmation in other populations.