AAC-11 is an antiapoptotic protein that is upregulated in most cancer cells. Increased expression of AAC-11 confers a survival advantage when cancer cells are challenged with various stresses and contributes to tumor invasion and metastases, whereas its deregulation reduces resistance to chemotherapeutic drugs. The antiapoptotic effect of AAC-11 may be clinically relevant as its expression correlates with poor prognosis in several human cancers. Thus, inactivation of AAC-11 might constitute an attractive approach for developing cancer therapeutics. We have developed an AAC-11-derived cell-penetrating peptide, herein named RT53, mimicking in part the heptad leucine repeat region of AAC-11, which functions as a protein-protein interaction module, and that can prevent AAC-11 antiapoptotic properties. In this study, we investigated the anticancer effects of RT53. Our results indicate that RT53 selectively kills cancer cells while sparing normal cells. RT53 selectively inserts into the membranes of cancer cells, where it adopts a punctate distribution and induces membranolysis and release of danger-associated molecular pattern molecules. Systemic administration of RT53 inhibited the growth of preexisting BRAF wild-type and V600E mutant melanoma xenograft tumors through induction of apoptosis and necrosis. Toxicological studies revealed that repetitive injections of RT53 did not produce significant toxicity. Finally, RT53-killed B16F10 cells induced tumor growth inhibition in immunocompetent mice following a rechallenge with live cancer cells of the same type. Collectively, our data demonstrate that RT53 possesses tumor-inhibitory activity with no toxicity in mice, suggesting its potential as a therapeutic agent for the treatment of melanoma and probably other cancers. Cancer Res; 76(18); 5479-90. ©2016 AACR.