Lichen sclerosus is a chronic relapsing disease, usually treated with ultra-potent corticosteroids. As immunological alterations are considered important aetiopathogenetic factors in lichen sclerosus, the new immunomodulating topical agents, such as tacrolimus and pimecrolimus, have been employed sporadically as alternative therapies. The aim of this study was to evaluate the therapeutic effects of tacrolimus 0.1% ointment in lichen sclerosus in 11 patients unresponsive or poorly responsive to previous treatments. Tacrolimus 0.1% ointment was applied twice daily for 6 weeks, then tapered over a further 6 weeks. Symptoms and objective parameters were evaluated and quantified at the start, after 6 weeks, at the end of the topical treatment, and at follow-up visits. Improvement or remission of symptoms was observed in the patients who completed the study, while objective parameters were poorly influenced and often were not related to symptom behaviour. Topical tacrolimus can be considered an alternative treatment for lichen sclerosus.