Phosphatidylinositol-3-kinases (PI3K) are a family of enzymes that act downstream of cell surface receptors leading to activation of multiple signaling pathways regulating cellular growth, proliferation, motility, and survival. To date, most research efforts have focused on a group of PI3K-family enzymes termed class I, of which the most studied member is PI3Kα. PI3Kα is an oncogene frequently mutated in human cancer, as is the chief negative regulator of the pathway, the tumor suppressor PTEN. Recently, it has been suggested that tumors deficient for PTEN might depend on the function of another class I member, PI3Kβ, to sustain their transformed phenotype. Taken together, these findings provide a significant medical rationale to study the signaling cascades regulated by PI3Kα and PI3Kβ particularly in the context of their role in the development and maintenance of human cancer. Here, we summarize the current understanding of the upstream receptor regulation of the two PI3K isoforms and their roles in cancer as well as their functional requirements in downstream signaling cascades.