The present study aimed to observe the cytotoxic activity of allogeneic natural killer (NK) cells on U251 glioma cells and to investigate their mechanism of action to establish an effective treatment strategy for neuroglioma. Cell survival curves, colony formation assays and karyotype analysis were performed to investigate the characteristics of U251 glioma cells. The present study demonstrated that natural killer group 2, member D (NKG2D)‑major histocompatibility complex class I‑related chain A/B (MICA/B) interactions contributed to the cytotoxic effect of NK cells on K562 and U251 cells. In antibody‑blocking assays to inhibit NKG2D ligands, the cytotoxic activity was not completely attenuated, which suggested that other signaling pathways contribute to the cytotoxic activity of NK cells on tumor cells in addition to the NKG2D‑mediated activity. The present study identified that the expression levels of NKG2D ligands on the surface of target cells influenced the strength of the NK cell immune response. Furthermore, allogeneic NK cells were observed to kill glioma cells in vitro, and this anticancer activity is associated with the rate of NKG2D expression on the surface of glioma cells.