AbstractTat, the transactivation factor of human immunodeficiency virus type 1 (HIV‐1), represents one of the major players mediating the loss of CD4‐positive T‐lymphocytes in HIV‐1‐infected patients, primarily due to the ability of Tat to trigger apoptosis. However, the molecular events underlying this process remain elusive. In this study, we provide evidence that Tat interacts with Eg5, a microtubule‐associated motor protein, and allosterically modulates the ATPase activity of Eg5 by affecting ADP release from the enzyme's active centre. This action of Tat impairs the formation of the mitotic spindle and activates the spindle checkpoint, thereby blocking cell cycle progression at mitosis and leading to apoptosis. Further studies reveal that lysine 85 in the carboxyl terminus of Tat is critical for its interaction with Eg5 and hence its effects on Eg5 activity, mitotic progression, and apoptosis. These findings identify Tat as a viral regulator of Eg5 and provide novel insights into the mechanisms of action of Tat in mediating the reduction of CD4‐positive T‐lymphocytes. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.