Abstract  This investigator-blinded randomized controlled trial was designed to determine whether tacrolimus ointment (Protopic, Fujisawa Healthcare) decreased the severity of localized lesions of canine atopic dermatitis (AD). Twenty dogs with AD were enrolled if they exhibited lesions on both front metacarpi. Each foot was randomized to be treated with 0.1% tacrolimus or placebo (vaseline) ointment twice daily for 6 weeks. Before, and every 2 weeks during the study, erythema, lichenification, oozing and excoriations each were graded on a 10-point scale (maximal total score: 40). The primary outcome measures were the percentage reduction from baseline of lesional scores and the number of subjects whose scores had decreased by 50% or greater at study end. Intention-to-treat analyses were used. At study onset, lesional scores were not significantly different between sites treated with tacrolimus or placebo. After 6 weeks, the percentage reduction from baseline scores was higher for tacrolimus-treated sites (median: 63%; 95% confidence interval: 39–67) than for placebo-treated feet (median: 3%; confidence interval: −2–13) (Wilcoxon test; P = 0.0003). When tacrolimus was applied, lesions decreased by 50% or greater in 15/20 dogs (75%); these dogs were those that completed the study. In contrast, this benchmark was not reached for any placebo-treated feet (Fisher's test; P < 0.0001). Adverse drug events consisted of minor irritation in some lesional areas treated with tacrolimus. Results of this trial suggest that the application of 0.1% tacrolimus ointment is useful for reducing the severity of localized skin lesions of canine AD.