Phosphoinositide 3-kinases (PI3Ks) are implicated in a broad spectrum of cellular activities, such as growth, proliferation, differentiation, migration, and metabolism. Activation of class I PI3Ks by mutation or overexpression correlates with the development and maintenance of various human cancers. These PI3Ks are heterodimers, and the activity of the catalytic subunits is tightly controlled by the associated regulatory subunits. Although the same p85 regulatory subunits associate with all class IA PI3Ks, the functional outcome depends on the isotype of the catalytic subunit. New PI3K partners that affect the signaling by the PI3K heterodimers have been uncovered, including phosphate and tensin homolog (PTEN), cyclic adenosine monophosphate-dependent protein kinase (PKA), and nonstructural protein 1. Interactions with PI3K regulators modulate the intrinsic membrane affinity and either the rate of phosphoryl transfer or product release. Crystal structures for the class I and class III PI3Ks in complexes with associated regulators and inhibitors have contributed to developing isoform-specific inhibitors and have shed light on the numerous regulatory mechanisms controlling PI3K activation and inhibition.