The stratum corneum of epidermis is an essential barrier against the external environment and water loss. This study aimed to develop an organotypic culture model that targets the reconstruction of the stratum corneum using canine keratinocyte-derived CPEK cells. The CPEK cells cultured at the air-liquid interface became stratified and formed a stratum corneum-like layer on stratum spinosum- and stratum granulosum-like layers. The CPEK cells in the stratum granulosum-like layer expressed the cornified cell envelope (CCE)-related proteins loricrin and keratinocyte differentiation-associated protein. Organotypically cultured CPEK cells were considered to form a CCE at the stratum granulosum-like layer, allowing the formation of a stratum corneum-like layer. The organotypic culture of CPEK cells could be useful for studying the barrier function of canine stratum corneum.