Darier's disease is an autosomal dominantly inherited skin disorder characterized by loss of adhesion between epidermal cells, breakdown of desmosome–keratin filaments, and abnormal keratinization. ATP2A2 has been identified as the causative gene of Darier's disease. This gene encodes the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) isoform 2 pump, which transports Ca²⁺ from the cytosol into the endoplasmic reticulum lumen to maintain a low cytosolic Ca²⁺ concentration. Using indirect immunofluorescence and biochemical analysis, we investigated the distribution of key desmosomal proteins in normal human and Darier's disease keratinocytes under various calcium conditions. We show that inhibition of SERCA by thapsigargin in normal human keratinocytes impairs the trafficking of the desmoplakins, desmoglein, and desmocollin to the cell surface; these proteins show a diffuse cytoplasmic distribution and, together with plakoglobin, form detergent-insoluble aggregates. In Darier's disease keratinocytes, only the trafficking of desmoplakin is significantly inhibited; in these cells, desmoplakin forms insoluble aggregates when extracted with mild detergent. In contrast, the transmembrane proteins desmoglein and desmocollin are efficiently transported to the cell surface. These proteins, along with plakoglobin, remain equally distributed between detergent-soluble and -insoluble fractions. We also demonstrate an interaction between SERCA2 and desmoplakin during differentiation. Our results provide further insights into the critical role of calcium ATPases in maintaining epidermal integrity.