Summary We describe a mechanism by which the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein is down-regulated to induce apoptosis. ARTS (Sept4_i2) is a tumor suppressor protein that promotes cell death through specifically antagonizing XIAP (X linked Inhibitor of Apoptosis). ARTS and Bcl-2 reside at the outer mitochondrial membrane in living cells. Upon apoptotic induction, ARTS brings XIAP and Bcl-2 into a ternary complex allowing XIAP to promote ubiquitylation and degradation of Bcl-2. ARTS binding to Bcl-2 involves the BH3 domain of Bcl-2. Lysine 17 in Bcl-2 serves as the main acceptor for ubiquitylation and a Bcl-2 K17A mutant has increased stability and is more potent in protection against apoptosis. Bcl-2 ubiquitylation is reduced in both XIAP and Sept4/ARTS deficient MEFs, demonstrating that XIAP serves as an E3-ligase for Bcl-2 and ARTS is essential for this process. Collectively, these results suggest a distinct model for the regulation of Bcl-2 by ARTS-mediated degradation.