Entry into mitosis in eukaryotes requires the activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 is opposed by protein phosphatases in two ways: They inhibit activation of Cdk1 by dephosphorylating the protein kinases Wee1 and Myt1 and the protein phosphatase Cdc25 (key regulators of Cdk1), and they also antagonize Cdk1's own phosphorylation of downstream targets. A particular form of protein phosphatase 2A (PP2A) containing a B55 delta subunit (PP2A-B55 delta) is the major protein phosphatase that acts on model CDK substrates in Xenopus egg extracts and has antimitotic activity. The activity of PP2A-B55 delta is high in interphase and low in mitosis, exactly opposite that of Cdk1. We report that inhibition of PP2A-B55 delta results from a small protein, known as alpha-endosulfine (Ensa), that is phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of PP2A-B55 delta. This pathway represents a previously unknown element in the control of mitosis.