Loss-of-function mutations in filaggrin gene are major predisposing factors for atopic dermatitis (AD). Although various reports suggest that filaggrin contributes stratum corneum (SC) barrier formation, the lack of filaggrin-null animal model has hampered progress to elucidate the pathogenic mechanism. Flaky tail / matted (ft/ma) mice have been used as a model of filaggrin deficiency, however these mice do not present complete loss of filaggrin and have unknown gene mutation “matted”. Therefore, we generated filaggrin-null (Flg-/-) mice and evaluated the impact of filaggrin loss in vivo. Flg-/- mice showed dry and scaly skin, but did not develop spontaneous dermatitis, in contrast to ft/ma mice. Despite of decreased natural moisturizing factor level in Flg-/- mice, which are degradation products of filaggrin, water content and transepidermal water loss were not affected in Flg-/- SC, indicating that hydration status of SC might not correlate with dry skin manifestation and natural moisturizing factor level. Histological observation suggested the premature detachment of cornified layer in Flg-/- mice, and tape stripping analysis demonstrated that Flg-/- SC were more fragile and more susceptible to physical stress. Loss of keratin pattern, which is important for maintaining SC integrity, was observed in Flg-/- SC using electron microscopy, and it is likely attributable to fragility in Flg-/- SC. Increased penetration of substances into SC was confirmed by permeability assay using liposome-encapsulated calcein. Barrier defect in Flg-/- mice led to enhanced hapten-induced contact hypersensitivity responses and humoral responses to topically immunized protein antigen, but the responses in ft/ma mice were constantly stronger, suggesting an additional role for matted. Our Flg-/- mice represent pure filaggrin deficiency, and should provide a valuable tool to dissect the function of SC, especially in the context of AD.