Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes extensively involved in cell signaling, acting downstream of tyrosine kinase and G-protein coupled receptors. Based on structural and substrate specificity, mammalian PI3Ks include class I (A, B), class II and class III enzymes. Several lines of evidence have shown that PI3Ks participate in the regulation of fundamental cellular functions such as cell growth, apoptosis, motility and vesicular trafficking, as well as in the modulation of physiological processes such as immunity, metabolism and cardiovascular physiology. Moreover, a deregulation of the PI3K pathway has been shown in several disease states such as cancer, inflammation and cardiovascular diseases, in human and animal models. Hence, the pharmacological inhibition of PI3Ks has emerged as a novel potential therapeutic option. Older and broad-spectrum PI3Ks inhibitors such as wortmannin and LY294002 have been useful tools for the elucidation of signaling pathways. However, isoform selectivity and adequate pharmacokinetic properties are now necessary for a selective therapeutic approach. Recently, the understanding of the crystallographic structure of PI3Ks has allowed the development of a multitude of inhibitors. In this review, we will present a general outline of PI3K functions in cellular, physiological and disease processes and discuss the main pharmacological compounds available for isoform-specific inhibition and the related evidence regarding their use in disease models.