Netherton syndrome (NTS) is a rare genetic skin disease caused by mutations in the serine protease inhibitor Kazal-type 5 gene, which encodes the lympho-epithelial Kazal-type-related inhibitor. NTS patients have profoundly impaired skin barrier function. As stratum corneum (SC) lipids have a crucial role in the skin barrier function, we investigated the SC lipid composition and organization in NTS patients. We studied the SC lipid composition by means of mass spectrometry, and the lipid organization was examined by infrared spectroscopy and X-ray diffraction. Decreased free fatty acid (FFA) chain length and increased levels of monounsaturated FFAs were observed in the SC of NTS patients compared with controls. Furthermore, the level of short-chain ceramides (CERs) was enhanced in NTS patients and a strong reduction in long-chain CER levels was seen in several patients. The changes in lipid composition modified the lipid organization leading to an increased disordering of the lipids compared with the controls. In addition, in a subgroup of patients the organization of the lipid layers changed dramatically. The altered FFA and CER profiles in NTS patients corresponded to changes in the expression of enzymes involved in SC lipid processing. The observed changes in lipid composition, lipid organization, and enzyme expression are likely to contribute to the barrier dysfunction in NTS.