Atopic dermatitis (AD) is a common pruritic skin disease associated with skin barrier defects and immune dysregulation. Conventional therapies for AD include topical treatments with corticosteroids and calcineurin inhibitors, which exert anti-inflammatory and immune regulatory effects. Thus, it would be important to identify a new drug that can effectively restore both skin barrier and immune abnormalities. We have reported a unique diet-induced AD mouse model. Hairless mice fed a special diet (named HR-AD) show AD-like pruritic dermatitis, which is caused mainly by deficiency of polyunsaturated fatty acids. The present study aimed to examine the effects of topical γ-linolenic acid (GLA) on AD symptoms in this model. Topical application of GLA suppressed Th2-mediated skin inflammation and itch-related scratching behavior. Furthermore, GLA treatment reduced transepidermal water loss with increasing covalently bound ceramides, which are essential for skin barrier formation. Genome-wide gene expression analysis revealed that GLA primarily regulated expression of epidermal differentiation genes, such as Sprr2d, and S100a8. Therefore, GLA may play various roles in skin barrier and immune regulation and could be a therapeutic candidate for AD.