Increased secretion of antimicrobial peptides and cytokines is present in atopic skin. The purpose of this study was to compare the production of β-defensin (cBD)3-like, cathelicidin (cCath) and cytokines in atopic and healthy canine keratinocytes. Seven atopic house dust mites (HDMs) sensitive and five healthy age-matched beagles were used. Keratinocytes were stimulated for 24 h, and the supernatant was collected. A significantly higher production of cBD3-like was present at baseline in atopic compared with healthy keratinocytes, but cBD3-like did not increase after stimulation. IL-17 and lipopolysaccharide increased cBD3-like in healthy compared with atopic keratinocytes. cCath increased in both groups after stimulation. Atopic keratinocytes exposed to HDM produced more IL-8 and keratinocyte-derived chemokine-like than healthy keratinocytes. Exposure to HDM induced an increased IL-8 in atopic keratinocytes and a decreased IFN-γ in healthy keratinocytes. These results may suggest an over sensitization of atopic keratinocytes and a possible impairment of the cutaneous defense against micro-organisms.