Single-chain Fv fragments (scFvs) consist of the variable heavy-chain (VH) and variable light-chain (VL) domains, which are the smallest immunoglobulin fragments containing the whole antigen-binding site. Human soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) proves to acquire a potent pro-apoptotic activity only after selective binding to a predefined tumor cell surface antigen and has no off-target effects towards normal cells. Glioblastoma multiforme (GBM) is the most frequent and aggressive type of brain tumor and overexpresses human multidrug resistance protein 3 (MRP3). In this study, we designed a novel fusion protein, termed scFvM58-sTRAIL, in which the MRP3-specific scFv antibody M58 was genetically fused to the N-terminus of human soluble TRAIL (sTRAIL). The recombinant scFvM58-sTRAIL fusion protein, expressed in Escherichia coli, was purified by chromatography and tested for cytotoxicity. scFvM58-sTRAIL showed a significant apoptosis-inducing activity towards MRP3-positive GBM cells in vitro. The pro-apoptotic activity of scFvM58-sTRAIL towards GBM cells was strongly inhibited in the presence of the parental scFvM58 antibody, suggesting that cytotoxic activity is MRP3-restricted. In a control experiment with MRP3-negative Jurkat cells, scFvM58-sTRAIL did not induce apparent apoptosis. In addition, through target antigen-restricted binding, scFvM58-sTRAIL was capable of activating not only TRAIL-R1 but also TRAIL-R2. In conclusion, our results suggest that fusion protein scFvM58-sTRAIL with specificity for MRP3 is a highly selective therapeutic agent and may provide an alternative therapy for human GBM.