Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by immune dysregulation with type 2 immune activation and impaired epidermal barrier function. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play critical roles in skin homeostasis and disease, yet the contribution of specific lncRNAs to AD pathogenesis remains largely unexplored. This study focuses on LINC01605, lncRNA, and its potential role in regulating keratinocyte function and contributing to AD. To characterize the expression and regulation of LINC01605, RNAscope in situ hybridization was performed on healthy and AD skin biopsies. Primary human keratinocytes were stimulated with type 2 cytokines (IL-4, IL-13, and IL-31) to assess cytokine-dependent regulation of LINC01605 expression. To study its functional role, siRNA-mediated knockdown of LINC01605 was carried out, followed by qPCR analysis of epidermal differentiation markers, dispase-based cell–cell adhesion assays, and immunofluorescence microscopy. To investigate downstream molecular pathways, RNA sequencing was performed on keratinocytes after LINC01605 depletion, followed by differential expression and Gene Ontology (GO) analysis. RNAscope analysis revealed widespread and elevated expression of LINC01605 in the epidermis of lesional AD skin compared to healthy controls. Cytokine stimulation experiments revealed that LINC01605 expression was upregulated in response to IL-4 and IL-13, particularly at later time points, while IL-31 induced an earlier increase in LINC01605 expression. Knockdown of LINC01605 in keratinocytes led to increased expression of terminal differentiation markers and enhanced intercellular adhesion, indicating a role in suppressing epidermal differentiation and barrier formation. Transcriptomic profiling revealed significant alterations in gene expression following LINC01605 knockdown, including downregulation of genes related to keratinocyte differentiation, epidermal development, and endoplasmic reticulum stress. GO analysis confirmed enrichment of pathways relevant to skin barrier function and inflammation. These findings suggest that LINC01605 may contribute to epidermal barrier dysfunction in AD and could serve as a novel target for therapeutic intervention.