The permeability barrier in the stratum corneum of the skin (skin barrier) prevents the entry of foreign substances such as pathogens, allergens, and toxic compounds and water loss. Ceramides, lipids abundant in the stratum corneum, are essential for the formation of the skin barrier. Free ceramides (non-protein-bound ceramides) are components of the multilayered lipid structures (lipid lamellae) that exist between corneocytes. They are classified into non-acylated ceramides (conventional ceramides) and acylceramides. Acylceramides play an important role in the formation and maintenance of lipid lamellae. Protein-bound ceramides, which covalently bind to Cys residues of corneocyte surface proteins), are components of the corneocyte lipid envelope (CLE). We recently revealed that human stratum corneum contain 23 classes and 1,581 species of ceramides (18 classes/1,327 species of free ceramides and 5 classes/254 species of protein-bound ceramides) by LC-MS/MS analysis). Patients with atopic dermatitis have reduced amounts of ceramides and altered ceramide class composition. Mutations in genes involved in the synthesis of acylceramides and protein-bound ceramides cause congenital ichthyosis in humans and neonatal lethality due to skin barrier abnormalities in mice. We have identified many genes involved in acylceramide production (ELOVL1, CYP4F22, PNPLA1, ABHD5 and FATP4) and elucidated the details of the acylceramide synthesis pathway). In this presentation, I will describe the following four topics: 1. the diversity of ceramides in human stratum corneum, 2. ceramide analysis by LC-MS/MS, 3. skin disorders (atopic dermatitis and ichthyosis) caused by changes in ceramide composition, 4. the molecular mechanism of acylceramide production and its importance in skin barrier formation.