Cancer, cardiovascular disease, and neurological disease are currently three top public health concerns in much of the world. One protein found deregulated in many of these diseases is Protein Phosphatase 2A (PP2A), a group of diverse heterotrimeric holoenzymes containing regulatory subunits which are responsible for substrate specificity. There is limited structural and biochemical information on PP2A holoenzyme-substrate interactions and no PP2A substrate binding consensus sequences are yet identified. The study here has an important focus on one family of PP2A regulatory subunits, the B’’/PR72 family, which is involved in several cellular processes and uniquely regulated by calcium binding. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here I report the crystal structure of the trimeric PR70 holoenzyme at a resolution of 2.4 Å and in vitro characterization of the PP2A-PR70 holoenzyme and its interaction with Cdc6. The holoenzyme structure reveals that one of the PR70 calcium-binding motifs directly contacts the scaffold subunit, resulting in the most compact scaffold subunit conformation among all known PP2A holoenzymes. In vitro biochemical analysis demonstrated that PR70 can enhance PP2A-mediated dephosphorylation of pCdc6; in contrast, the B’ family of holoenzymes barely have any phosphatase activity on pCdc6, likely due to steric hindrance by a B’ loop near the active site. PR70 binds distinctively to the catalytic subunit near the active site, and this interaction is required for PR70 to enhance phosphatase activity toward Cdc6. These studies provide a structural basis for unique regulation of B&rsquo:’/PR72 holoenzymes by calcium ions, and suggest mechanisms for precise control of substrate specificity among PP2A holoenzymes. The study above suggests that local structural features and global conformational changes regulate substrate binding. As such, PP2A substrate recognition is dictated by three-dimensional architecture of holoenzymes, and PP2A substrates might not have consensus recognition sequences for holoenzyme binding. The study above also demonstrates how peptide-based assays help characterize PP2A substrate interactions. Given the vast amount of processes and pathways in which PP2A is involved, novel high-throughput methods are discussed for efficient characterization of PP2A substrates in vitro.