While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3'-kinase C2beta (PI3K-C2beta) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2beta-AKT survival pathway. ITSN associated with PI3K-C2beta on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2beta activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2beta and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.