Topical glucocorticoid (GC) is commonly applied in atopic dermatitis treatment. However, the chronic use of GC may be associated with significant side effects. In this study, we investigated whether long-term epicutaneous application of GC modulates scratching behaviour in dinitrofluorobenzene (DNFB) contact-sensitized mice. After challenge with DNFB, scratching behaviour was increased in DNFB-sensitized mice treated with GC in contrast to control mice. In addition, reverse transcriptase-polymerase chain reaction analysis demonstrated that the expression of preprotachykinin-A (PPT-A) mRNA, a precursor of substance P (SP), and inducible nitric oxide synthase (iNOS) mRNA in mice, to which GC was applied, was only observed. In order to evaluate the factors responsible for the augmented scratching behaviour, we injected various cytokines (interleukin-1alpha (IL-1alpha), IL-2, IL-3 and tumour necrosis factor-alpha (TNF-alpha)) subcutaneously into the ear of DNFB contact-sensitized mice before DNFB challenge. Among the cytokines, only IL-3 and TNF-alpha significantly increased scratching behaviour in DNFB contact dermatitis mice. Furthermore, PPT-A mRNA was only expressed in mice pre-injected with IL-3 before challenge, but not in those pre-injected with other cytokines. Taken together, our results suggest that topical GC may augment the itching sensation in DNFB-sensitized mice through modulation of iNOS and SP induced by IL-3.