The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent, which kills cancer cells selectively, while leaving normal cells unharmed. However, the emerging resistance of tumor cells and patients to TRAIL-induced apoptosis limits its further application. In this study, we developed a chimeric protein Annexin V-TRAIL (designated as TP8) with higher efficacy than TRAIL both in vitro and in vivo. In vitro, the EC50 of TP8 on a series of tumor cells was much lower than wild-type TRAIL. Annexin V provided this recombinant protein with higher efficacy, while leaving tumor specificity of TRAIL unchanged since TP8 had no effects on normal cells. In vivo, TP8 effectively suppressed tumor growth and prolonged tumor doubling time and tumor growth delay time in mouse xenografts involving multiple cancer cell types including A549, Colo205 and Bel7402. This study provides a new rational strategy to treat TRAIL-resistant cancers.