Purpose: XIAP [X-linked inhibitor of apoptosis (IAP) protein] is the best characterized mammalian caspase inhibitor. XIAP is frequently overexpressed in a variety of human tumors, and genetic inactivation of XIAP in mice protects against lymphoma. Therefore, XIAP is an attractive target for anticancer therapy. IAP antagonists based on a conserved IAP-binding motif (IBM), often referred to as "Smac-mimetics," are currently being evaluated for cancer therapy in the clinic. ARTS (Sept4-i2) is a mitochondrial proapoptotic protein which promotes apoptosis by directly binding and inhibiting XIAP via a mechanism that is distinct from all other known IAP antagonists. Here, we investigated the ability of peptides derived from ARTS to antagonize XIAP and promote apoptosis in cancer cell lines. Experimental Design: The ability of synthetic peptides, derived from the C-terminus of ARTS, to bind to XIAP, stimulate XIAP degradation, and induce apoptosis was examined. We compared the response of several cancer cell lines to different ARTS-derived peptides. Pull-down assays were used to examine binding to XIAP, and apoptosis was evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, caspase activation, and Western blot analyses of caspase substrates. Results: The C-terminus of ARTS contains a unique sequence, termed ARTS-IBM (AIBM), which is important for binding to XIAP and cell killing. AIBM peptides can bind to XIAP-BIR3, penetrate cancer cells, reduce XIAP levels, and promote apoptosis. Conclusions: Short synthetic peptides derived from the C-terminus of ARTS are sufficient for binding to XIAP and can induce apoptosis in cancer cells. These results provide proof-of-concept for the feasibility of developing ARTS-based anticancer therapeutics.