High-grade non-muscle-invasive bladder cancer is commonly treated with Bacillus Calmette-Guérin, an immunotherapeutic that depends on fibronectin and tumor cell integrin α5β1 for internalization into bladder cancer cells. We previously showed that the anti-angiogenic peptide CLT1 forms cytotoxic complexes with fibronectin that are cooperatively internalized into proliferating endothelium through ligation of integrins and chloride intracellular channel 1. While CLT1 has no effect on mature, differentiated cells, we show here that CLT1 is highly cytotoxic for a panel of bladder tumor cell lines as well as a variety of cell lines derived from kidney, lung, breast, and prostate cancer. Paralleling our previous results, we found CLT1-induced tumor cell death to be increased in the presence of fibronectin, which mediated CLT1 internalization and subsequent autophagic cell death in a mechanism that depends on tumor cell integrin α5β1 and chloride intracellular channel 3 (CLIC3). This mechanistic link was further supported by our results showing upregulation of α5β1 and CLIC3 in CLT1-responsive tumor cell lines and colocalization with CLT1 in tumor tissues. Incubating tumor tissue from patients with bladder cancer with fluorescein-conjugated CLT1 resulted in a strong and specific fluorescence whereas normal bladder tissue remained negative. On the basis of its affinity for bladder tumor tissue and strong antitumor effects, we propose that CLT1 could be useful for targeting bladder cancer.