Background: We investigated whether topical administration of melatonin ameliorates radiation-induced skin fibrosis (RISF) and inhibits the expression of profibrogenic genes in mice. Materials and Methods: Forty-eight female BALB/c mice were randomly divided into three groups: topically applied 5% ethanol (Control), topically applied 5% ethanol plus irradiation (IR), and topically applied melatonin plus irradiation (Mel+IR). The right hind legs of the IR and Mel+IR group mice were exposed to two fractions of electron beam radiation (20 Gy × 2). For 4 weeks, melatonin solution (10 mg/day) was topically applied to Mel+IR group mice. Fourteen days after IR, the relative levels of transforming growth factor (TGF)-β1 mRNA expression and TGF-β1 protein in skin specimens were analyzed by real-time quantitative PCR and immunohistochemical staining. Dermal thickness and tissue collagen accumulation were measured at 100 days post irradiation. Results: The Radiation caused a 2.2-fold increase in TGF-β1 mRNA expression relative to that in control group, which was decreased by 37% following melatonin treatment (P = 0.024). We also observed substantial reduction of TGF-β1 expression in immunohistochemical studies. The mean values of dermal thickness were 105 ± 11 μm (Control), 195 ± 21 μm (IR), and 148 ± 19 μm (Mel+IR). Dermal thickness and collagen accumulation, which increased in the IR group, was significantly reduced by topically applied melatonin.  Conclusion: Topical administration of melatonin successfully attenuated RISF.