Lipid rafts are cholesterol-rich cell signaling platforms, and their physiological role can be explored by cholesterol depletion. To characterize transcriptional changes ongoing after lipid raft disruption in epidermal keratinocytes, a cell type that synthesizes its cholesterol in situ, we performed whole-genome expression profiling. Microarray results show that over 3,000 genes are differentially regulated. In particular, IL-8, urokinase-like plasminogen activator receptor, and metalloproteinases are highly upregulated after cholesterol extraction. Quantitative reverse transcriptase PCR validation and protein release measurements demonstrate the physiological relevance of microarray data. Major enriched terms and functions, determined by Ingenuity Pathways Analysis, identify cholesterol biosynthesis as a major function, illustrating the specificity of keratinocyte response toward cholesterol depletion. Moreover, the inflammatory skin disorder atopic dermatitis (AD) is identified as the disease most closely associated with the profile of lipid raft-disrupted keratinocytes. This finding is confirmed in skin of AD patients, in whom transcript levels of major lipid raft target genes are similarly regulated in lesional atopic skin, compared with non-lesional and normal skin. Thus, lipid raft disruption evokes typical features of AD, thereby suggesting that lipid raft organization and signaling could be perturbed in atopic keratinocytes.