The Retinoblastoma Protein (Rb) is a sentinel of the cell division process. When phosphorylated, Rb is inactivated and physically releases its protein-binding partner, E2F; a potent transcription factor that up-regulates many of the genes required for DNA synthesis and cell division. Aberrant deregulation of Rb, either through the loss of functional Rb or the persistence of hyper-phosphorylated Rb, is a common event in the development and progression of cancer. This study uses protein X-ray crystallography, NMR, ITC and SAXS to characterize three distinct structural changes to Rb that are caused by certain phosphorylation events. Specifically, we find phosphorylation of S608 induces the formation of a helix within the pocket linker region of Rb, which competitively inhibits E2F-binding. T373 phosphorylation promotes a docking interaction between Rb's two structured domains; this causes an allosteric change to a critical E2F-binding surface of Rb and dramatically weakens the Rb-E2F interaction. Lastly, phosphorylation of Rb's C-terminus at S788 and S795 also promotes an intramolecular interaction that destabilizes the Rb-E2F complex. These three distinct phosphorylation-induced mechanisms provide supporting evidence for structure-based theories of how Rb phosphorylation promotes the release and activation of E2Fs. A knowledge of these mechanisms is the first step in understanding how Rb may be therapeutically targeted to regain its function in cancer cells.