Atopic dermatitis (AD) is a multifactorial chronic inflammatory skin disease characterized by skin barrier dysfunction, eczematous lesions, pruritus, and abnormal immune responses. In this study, we assessed the therapeutic effect of topical applied conjugated linoleic acid (CLA) on a murine AD model that was developed by repetitive applications of 2, 4-dinitrofluorobenzene (DNFB). 2% or 5% CLA could markedly ameliorate AD-like skin lesions, scratching behaviour and skin inflammation as evidenced by the reduced inflammatory blood cells, IgE and Th2-related cytokine levels, and the infiltration of mast cells and inflammatory cells to the dermal tissues. Moreover, topical application with CLA modulated skin barrier repair including maintaining a balanced skin pH and increasing skin hydration, partially mediated by upregulating skin barrier-related protein, filaggrin (FLG). In addition, topical CLA significantly dose-dependently inhibited pro-inflammatory cytokines including interleukin (IL)-6, IL-1β, tumour necrosis factor (TNF)-α and pro-inflammatory enzyme expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in inflamed mice skin. Its anti-inflammatory effect was associated with the inhibition of DNFB-stimulated IκBα and NF-κB p65 phosphorylation in mouse skin. Taken together, our results suggest that locally applied CLA exerts potentially protective effects against AD lesional skin at least in part, due to regulation of skin barrier function and inflammatory response.