Atopic eczema (AE) is a chronic relapsing inflammatory skin disease affecting currently over 15% of Caucasian children and 2–10% of adults. Patients suffer from an impaired skin barrier function, which is located primarily in the stratum corneum (SC). In particular lipids in the SC provide the barrier, and protect the body against environmental pathogens and regulate transepidermal water loss. The lipids are predominantly composed of cholesterol, free fatty acids and ceramides (CERs). The latter class consists of multiple subclasses and each subclass shows variation in carbon chain length. Several studies have demonstrated changes in CER subclasses, but no detailed study is reported on the effect of chain length distribution of CERs on the skin barrier. We report a case control study in which the CER composition in SC of non-lesional AE skin has been examined. Drastic changes are observed in CER composition, in particular in the chain length distribution of the CERs: Both the increased presence of extremely short CERs as well as a decrease in very long CERs contribute to a reduction in CER chain length in AE patients compared to the control group. This reduction in chain length showed an excellent correlation to a reduced barrier function and disease severity. The results were independent of any filaggrin mutation, suggesting that the role of lipids is not related to the effects of filaggrin on the SC barrier function. The results demonstrate for the first time that CER chain length is an important factor in skin barrier dysfunction in AE, and envisage that these new insights provide a new entry in therapy and prevention of AE.