Mitochondria fulfill essential roles in ATP production, metabolic regulation, calcium signaling, generation of reactive oxygen species (ROS) and additional determinants of cellular health. Recent studies have highlighted a role for mitochondria during cell differentiation, including in skin epidermis. Furthermore, the observation of oxidative stress in keratinocytes from Krt16 null mouse skin, a model for pachyonychia congenita (PC) -associated palmoplantar keratoderma, prompted us to examine the role of K16 protein and its partner K6 in regulating the structure and function of mitochondria. Electron microscopy revealed major anomalies in mitochondrial ultrastructure in late stage, E18.5, Krt6 null embryonic mouse skin. Follow-up studies utilizing biochemical, metabolic, and live imaging readouts showed that, relative to controls, skin keratinocytes null for Krt6 or Krt16 exhibit elevated ROS, reduced mitochondrial respiration along with significant alterations in the length, intracellular distribution and movement of mitochondria within the cell. These findings highlight a novel role for K6 and K16 in regulating mitochondrial morphology, dynamics and function and shed new light on the causes of oxidative stress observed in PC and related keratin-based skin disorders.