DNA demethylation has been primarily studied in the context of development biology, cell fate, and cancer, with less attention on inflammation. In this article, we investigate the association between DNA methylation and production of the chemoattractant cytokine eotaxin-3 in the tissue of patients with allergic disease. Regions of the human eotaxin-3 promoter were found to be hypomethylated in primary epithelial cells obtained from allergic tissue compared with normal control tissue. The demethylation of a specific CpG site (designated CpG 2), which is juxtaposed to a key cAMP-responsive element site, was significantly demethylated in patient-derived compared with normal control tissue-derived epithelial cells. Levels of methylation at CpG 2 inversely correlated with basal and IL-13-induced eotaxin-3 gene expression. Conversely, global inhibition of methylation with 5-azacytidine promoted eotaxin-3 production in association with decreasing CpG 2 methylation. In addition, the basal and IL-13-induced eotaxin-3 transcriptional activity was suppressed by promoter methylation using a methylation-free in vitro system. Furthermore, EMSAs demonstrated that the attachment of CREB binding protein and activating transcription factor 2 (ATF-2) to the cAMP-responsive element site was methylation dependent. Taken together, these data identify a contributory role for DNA methylation in regulating eotaxin-3 production in human allergic inflammation.