Error-free genome duplication and segregation are ensured through the timely activation of ubiquitylation enzymes. The anaphase-promoting complex or cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, is regulated by phosphorylation. However, the mechanism remains elusive. Using systematic reconstitution and analysis of vertebrate APC/Cs under physiological conditions, we show how cyclin-dependent kinase 1 (CDK1) activates the APC/C through coordinated phosphorylation between Apc3 and Apc1. Phosphorylation of the loop domains by CDK1 in complex with p9/Cks2 (a CDK regulatory subunit) controlled loading of coactivator Cdc20 onto APC/C. A phosphomimetic mutation introduced into Apc1 allowed Cdc20 to increase APC/C activity in interphase. These results define a previously unrecognized subunit-subunit communication over a distance and the functional consequences of CDK phosphorylation. Cdc20 is a potential therapeutic target, and our findings may facilitate the development of specific inhibitors.