Peptide-based therapies have emerged as one of the most promising therapeutics strategy in cancer-targeted therapy. Using our laboratory newly identified peptide TMTP1 and diphtheria toxin, we developed a new fusion protein that showed remarkable ability to target highly metastatic tumors. Fusion protein toxins were generated by fusing the first 390 amino acids of diphtheria toxin [truncated diphtheria toxin (DT390)] to different repeats of peptide TMTP1 (DT390-TMTP1, DT390-biTMTP1, and DT390-triTMTP1). Efficacies of the recombinant fusion proteins on tumor growth and metastasis were evaluated in vitro and in vivo. DT390-triTMTP1 showed the most powerful toxicity against cancer, which led to tumor growth retardation or regression and prolonged survival of human prostate cancer PC-3M-1E8 subcutaneously bearing or gastric cancer MKN-45 orthotopic nude mice. Increased TUNEL and caspase-3 staining and reduced ki67 staining in tumor cells suggested that the anticancer effects of DT390-triTMTP1 were through selectively inducing apoptosis and inhibiting proliferation of cancer cells. In a murine model of human orthotopic gastric carcinoma, DT390-biTMTP1 significantly inhibited metastases to liver and spleen, while DT390-triTMTP1 not only totally suppressed metastasis but also reduced primary tumors by 66.6%. In the biodistribution test, DT390-triTMTP1 was observed to home to tumor tissue rapidly and lasted over 48 h, with only a transient appearance in liver and kidney immediately after injection. Thus, our present study provided a novel recombinant fusion protein DT390-triTMTP1 with preferential targeting and high cytotoxicity, which may be a promising strategy for the targeted therapy of cancer metastasis.