The retinoblastoma tumor suppressor (Rb) is a multifunctional protein that primarily regulates the cell cycle but also has roles in cellular differentiation, DNA damage response, and apoptosis. The loss of Rb is a key event in the development or progression of many cancers. Essential functions of Rb occur through its pocket domain, which is necessary for regulating binding interactions with E2F transcription factors and transcription repressors. The pocket domain is the most highly conserved region of the multidomain protein, as well as the most frequent site of mutations. To understand what effects cancer missense mutations have on Rb’s pocket domain, we quantified functional changes caused by 75 different cancer-associated missense variants to Rb. We find that 43% of the missense variants tested reduce Rb-E2FTD binding and that many of these variants further destabilize the fold of the protein and show temperature-sensitive binding effects. Protein X-ray crystallography of four missense variants reveals how mutations structurally destabilize the protein fold and inhibit functional binding interactions. Taken together, this work provides the first understanding of the multiple ways through which stability, structure, and function of Rb’s pocket domain are altered by common missense mutations seen in cancer.