Epidermal keratinocytes have the potential to produce inflammatory mediators that are considered to play an important role in skin diseases such as atopic dermatitis (AD). Thus, cell lines of canine epidermal keratinocytes are useful for studying the biological reactivity of keratinocytes in vitro. However, there has been no report on properly analyzing the phenotype of canine keratinocyte cell lines. In this work, we performed phenotypic analysis of CPEK, which was derived from the epidermis of an adult dog in order to examine the phenotypic similarity with epidermal keratinocytes. The present findings indicated that CPEK cells expressed markers for epidermal keratinocytes including cytokeratin 14, alpha6 integrin and PCNA. Our findings demonstrated that CPEK could be a useful cell line for investigating the central role of epidermal keratinocytes in the pathogenesis of AD in vitro.