Objectives Occupational pesticide exposure is associated with a wide range of diseases, including lung diseases, but it is largely unknown how pesticides influence airway disease pathogenesis. A potential mechanism might be through epigenetic mechanisms, like DNA methylation. Therefore, we assessed associations between occupational exposure to pesticides and genome-wide DNA methylation sites. Methods 1561 subjects of LifeLines were included with either no (n=1392), low (n=108) or high (n=61) exposure to any type of pesticides (estimated based on current or last held job). Blood DNA methylation levels were measured using Illumina 450K arrays. Associations between pesticide exposure and 420 938 methylation sites (CpGs) were assessed using robust linear regression adjusted for appropriate confounders. In addition, we performed genome-wide stratified and interaction analyses by gender, smoking and airway obstruction status, and assessed associations between gene expression and methylation for genome-wide significant CpGs (n=2802). Results In total for all analyses, high pesticide exposure was genome-wide significantly (false discovery rate P<0.05) associated with differential DNA methylation of 31 CpGs annotated to 29 genes. Twenty of these CpGs were found in subjects with airway obstruction. Several of the identified genes, for example, RYR1, ALLC, PTPRN2, LRRC3B, PAX2 and VTRNA2-1, are genes previously linked to either pesticide exposure or lung-related diseases. Seven out of 31 CpGs were associated with gene expression levels. Conclusions We show for the first time that occupational exposure to pesticides is genome-wide associated with differential DNA methylation. Further research should reveal whether this differential methylation plays a role in the airway disease pathogenesis induced by pesticides.