As in humans, there is mounting evidence in support of an abnormal skin barrier contributing to the pathogenesis of canine atopic dermatitis (AD). Studies in people with AD have associated an abnormal skin barrier with deficiencies in ceramides, which represent important components of the stratum corneum (SC) intercellular lipid lamellae. Therefore, the goal of this study was to determine if the SC of dogs with AD is deficient in ceramides compared to normal dogs. Samples of SC were obtained from nonlesional skin of the caudal abdomen of 14 patients with AD and 14 age-, breed- and sex-matched healthy controls using a cyanoacrylate stripping procedure, and the subclass and relative amount of ceramides were assessed blindly by thin layer chromatography. Paired t-tests using R statistical computer software revealed the percentage amounts of ceramides 1 and 9 were significantly lower in nonlesional skin of AD dogs compared to controls (P = 0.034 and P = 0.047, respectively), and the cholesterol percentage amount was significantly higher in AD dogs than in controls (P = 0.016). Furthermore, the cholesterol/ceramide ratio was significantly higher in the AD group with respect to controls (P = 0.014). These findings suggest that decreased amounts of ceramides in the skin of dogs with AD may be involved in the impaired barrier function of their skin.