The overall prognosis of conventional chemotherapy for the treatment of bladder cancer is poor and reduction of its systemic side effects remains an unsolved issue. Targeted therapy for bladder cancer could improve therapeutic efficacy and reduce side effects. This study investigated a hybrid peptide (named Bld-1-KLA) composed of the CSNRDARRC peptide (Bld-1), which binds to bladder tumor cells, and the d-KLAKLAKKLAKLAK (KLA) peptide, which disrupts mitochondrial membrane and induces apoptotic cell death, as a bladder cancer-targeted therapeutic agent. Bld-1-KLA selectively bound to HT1376 bladder tumor cells and efficiently internalized into the cells but not to other types of tumor and normal cell lines. Bld-1-KLA exerted cytotoxic effects selectively to HT1376 cells (LC50 = 41.5 μM), but not to other types of cells. Pretreatment of cells with Bld-1 inhibited the binding and cytotoxicity by Bld-1-KLA in HT1376 cells. It induced apoptosis of bladder tumor cells, while Bld-1 or KLA alone showed much lesser effect on apoptosis, and was co-localized in mitochondria. Bld-1-KLA was stable up to 24 h in serum. In vivo fluorescence imaging showed that homing of Bld-1-KLA in the tumor in HT1376 tumor-bearing nude mice was greater than that of the control peptide-KLA after intravenous injection. Treatment of tumor-bearing mice with Bld-1-KLA, compared to the control peptide-KLA, induced apoptosis of tumor cells and inhibited tumor growth more efficiently. No significant side effects on body weight and the liver and myeloid function were observed in mice treated with Bld-1-KLA. These results suggest that Bld-1-KLA is a promising therapeutic agent for targeted therapy of bladder cancer.