Keratin 9 (K9) is a type I intermediate filament protein whose expression is confined to the suprabasal layers of palmoplantar epidermis. Though mutations in the K9 gene are known to cause epidermolytic palmoplantar keratoderma (EPPK), a rare dominant-negative skin disorder, its functional significance is poorly understood. To gain insight into the physical requirement and importance of K9, we generated K9-deficient (Krt9(-/-)) mice. Here, we report that adult Krt9(-/-) mice develop calluses marked by hyperpigmentation that are exclusively localized to the stress-bearing footpads. Histological, immunohistochemical and immunoblot analyses of these regions revealed hyperproliferation, impaired terminal differentiation and abnormal expression of keratins K5, K14 and K2. Furthermore, the absence of K9 induces the stress-activated keratins K6 and K16. Importantly, mice heterozygous for the K9-null allele (Krt9(+/-)) show neither an overt nor histological phenotype, demonstrating that one Krt9 allele is sufficient for developing normal palmoplantar epidermis. Together, our data demonstrates that the complete ablation of K9 is not tolerable in vivo and that K9 is required for terminal differentiation and maintaining the mechanical integrity of palmoplantar epidermis.Journal of Investigative Dermatology accepted article preview online, 20 August 2013. doi:10.1038/jid.2013.356.