The cell cycle is exquisitely controlled by multiple sequential regulatory inputs to ensure fidelity. Here we demonstrate that the final step in division, the physical separation of daughter cells, is controlled by a member of the PKC gene superfamily. Specifically, we have identified three phosphorylation sites within PKCɛ that control its association with 14-3-3. These phosphorylations are executed by p38 MAP kinase (Ser 350), GSK3 (Ser 346) and PKC itself (Ser 368). Integration of these signals is essential during mitosis because mutations that prevent phosphorylation of PKCɛ and/or PKCɛ binding to 14-3-3 also cause defects in the completion of cytokinesis. Using chemical genetic and dominant-negative approaches it is shown that selective inhibition of PKCɛ halts cells at the final stages of separation. This arrest is associated with persistent RhoA activation at the midbody and a delay in actomyosin ring dissociation. This study therefore identifies a new regulatory mechanism that controls exit from cytokinesis, which has implications for carcinogenesis.