DNA codes for genetic information. Furthermore, recent findings suggest that DNA offers additional function, particularly in the recognition of microorganisms. In this study, we investigated two classes of oligodeoxynucleotides (ODN) in skin keratinocytes; namely, an ODN comprising two cytidine-phosphate-guanosine (CpG) motifs (CpG-1-phosphorothioate (PTO)) and a poly-cytidine (Non-CpG-5-PTO) as control. Both fluorescence-tagged ODN were rapidly taken up by cells and accumulated already after 5 minutes in perinuclear compartments. In order to test whether ODN convey immunological effects in keratinocytes, secretion of IL-8 was measured. Interestingly, both CpG-1-PTO and Non-CpG-5-PTO suppressed basal and tumor necrosis factor alpha-induced IL-8 levels measured in cell culture supernatants. Experiments using deletion mutant revealed a critical length of approximately 16 nucleotides conveying IL-8 suppression. Studies regarding the ODN backbone offered that PTO bondings are critical for significant IL-8 suppression. In order to substantiate the anti-inflammatory response, a contact hypersensitivity mouse model was utilized. Topical application of Non-CpG-5-PTO-containing ointments reduced ear thickness in sensitized mice. Taken together, these findings suggest an anti-inflammatory effect of ODN in epithelial cells in vitro and in vivo, indicating that DNA molecules offer distinct biological activities restricted to the physiological compartment applied. This effect seems to be independent from Toll-like receptor 9.