Retinoblastoma protein (RB) prevents cell cycle progression by binding to and inhibiting E2F transcription factors. This interaction is regulated by cyclin-dependent kinase (CDK), which phosphorylates RB, thereby inactivating it. RB phosphorylation at Thr373 and Ser608 has previously been shown to inhibit the association of the RB pocket domain with the E2F transactivation domain (TD). This study elucidates the mechanism by which this occurs by delineating the structural changes induced by Ser608 and Thr373 phosphorylation. Although both phosphorylation events ultimately prevent the association of the RB pocket domain with E2F, they stimulate distinct structural changes: Ser608 phosphorylation induces a conformational change in the large loop within the RB pocket domain that blocks binding of E2F TD; and Thr373 phosphorylation triggers a conformational change that results in docking of the RB pocket and the RB amino-terminal domain.