There is a lack of studies on the effect of tacrolimus on eosinophils and related molecules including eotaxin, CCR3, RANTES and interleukin (IL)-5. To investigate the effects of tacrolimus on in vivo eosinophil counts and on the related molecules eotaxin, CCR3, RANTES and IL-5 in patients with atopic dermatitis (AD). Lesional skin specimens and sera were obtained from 15 patients with AD and from 15 normal controls. For 8 weeks, the patients with AD applied 0.03% tacrolimus ointment to all affected areas twice daily. Blood sampling and skin biopsies were then repeated. We evaluated serum eotaxin and IL-5 levels, and tissue eotaxin, CCR3, RANTES and IL-5 levels. Additionally, tissue levels of eotaxin and CCR3 mRNA were measured. After treatment with topical tacrolimus twice daily for 8 weeks, significant decreases were found in serum IL-5 levels, immunoreactive cell counts of eotaxin, IL-5, CCR3 and RANTES in AD skin, and tissue eosinophil counts. However, the change in the serum eosinophil count was not statistically significant, and mRNA levels of eotaxin and CCR3 were not decreased significantly after treatment. Topical tacrolimus reduces the number of eosinophils in tissue and suppresses the expression of eotaxin, CCR3, RANTES and IL-5 related to proliferation, recruitment, activation and survival of eosinophils.