Cell–extracellular matrix (ECM) detachment is known to decrease extracellular signal–regulated kinase (ERK) signaling, an intracellular pathway that is central for control of cell behavior. How cell–ECM detachment is linked to downregulation of ERK signaling, however, is incompletely understood. We show here that focal adhesion protein Ras Suppressor 1 (RSU1) plays a critical role in cell–ECM detachment induced suppression of ERK signaling. We have identified prohibitin 2 (PHB2), a component of membrane lipid rafts, as a novel binding protein of RSU1, and mapped a major RSU1-binding site to PHB2 amino acids 150 to 206 in the C-terminal region of the PHB/SPFH (stomatin/prohibitin/flotillin/ HflKC) domain. The PHB2 binding is mediated by multiple sites located in the N-terminal leucine-rich repeat region of RSU1. Depletion of PHB2 suppressed cell–ECM adhesion–induced ERK activation. Furthermore, cell–ECM detachment increased RSU1 association with membrane lipid rafts and interaction with PHB2. Finally, knockout of RSU1 or inhibition of RSU1 interaction with PHB2 by overexpression of the major RSU1-binding PHB2 fragment (amino acids 150–206) effectively suppressed the cell–ECM detachment induced downregulation of ERK signaling. Additionally, expression of venus-tagged wild-type RSU1 restored ERK signaling, while expression of venus-tagged PHB2-binding defective RSU1 mutant in which the N-terminal leucine-rich repeat region is deleted did not. Taken together, Our findings identify a novel RSU1-PHB2 signaling axis that senses cell–ECM detachment and links it to decreased ERK signaling.