The human papillomavirus (HPV) E1 empty set E4 protein is the most abundantly expressed viral protein in HPV-infected epithelia. It possesses diverse activities, including the ability to bind to the cytokeratin network and to DEAD-box proteins, and in some cases induces the collapse of the former. E1 empty set E4 is also able to prevent the progression of cells into mitosis by arresting them in the G(2) phase of the cell cycle. In spite of these intriguing properties, the role of this protein in the life cycle of the virus is not clear. Here we report that after binding to and collapsing the cytokeratin network, the HPV type 16 E1 empty set E4 protein binds to mitochondria. When cytokeratin is not present in the cell, E1 empty set E4 appears associated with mitochondria soon after its synthesis. The leucine cluster within the N-terminal portion of the E1 empty set E4 protein is pivotal in mediating this association. After the initial binding to mitochondria, the E1 empty set E4 protein induces the detachment of mitochondria from microtubules, causing the organelles to form a single large cluster adjacent to the nucleus. This is followed by a severe reduction in the mitochondrial membrane potential and an induction of apoptosis. HPV DNA replication and virion production occur in terminally differentiating cells which are keratin-rich, rigid squamae that exfoliate after completion of the differentiation process. Perturbation of the cytokeratin network and the eventual induction of apoptotic properties are processes that could render these unyielding cells more fragile and ease the exit of newly synthesized HPVs for subsequent rounds of infection.