Glioblastoma (GBM) is a lethal brain tumor with limited therapeutic options. Bi-specific killer cell engagers (BiKEs) are novel immunotherapies designed to engage natural killer (NK) cells against cancer. We designed a BiKE molecule consisting of a single-domain CD16 antibody, an interleukin-15 linker, and a single-chain variable antibody against the glioma-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2). Recombinant BiKE protein was expressed in HEK cells and purified. Flow cytometric analysis of co-cultures of peripheral blood-derived NK cells with GBM6 and GBM39 patient-derived xenograft lines revealed significantly increased activation of NK cells (CD25+CD69+) and increased glioma cell killing following BiKE treatment compared to controls (n = 4,p< 0.01). Glioma cell killing was also confirmed via immunofluorescence staining for cleaved caspase-3 (p< 0.05). In vivo, intracranial delivery of NK cells with BiKE extended median survival in mice bearing GBM6 (p< 0.01) and GBM12 (p< 0.01) tumors compared to controls. Finally, histological analysis of brain tissues revealed a higher frequency of peritumoral NK cells in mice treated with BiKE than with NK cells alone (p< 0.05). In conclusion, we demonstrate that a BiKE generated in a mammalian expression system is functional in augmenting NK cell targeting of IL13Rα2-positive gliomas.