Osteosarcoma is a serious malignancy in pediatric patients, which comprises 2.4% of fatal cancer in children and achieves 20% of all primary bone cancers. In the present study, we employed three human osteosarcoma cell lines MG‑63, HOS and U2OS for susceptibility to cytolytic activity of freshly isolated healthy donor NK cells. Cells were lysed by NK cells in a dose dependent manner. MG‑63 cells exhibited less susceptibility to NK cells than HOS and U2OS cells at all cell ratios. The specific mechanism underlying the effects of NK cells on osteosarcoma cells was determined by antibody blockage experiments. The results revealed that granzyme B was the key factor in the NK cell‑induced cytotoxicity of human osteosarcoma cells. To the best of our knowledge, the present study is the first to investigate the expression of PD‑L1 in MG‑63, HOS and U2OS cells. The relative expression of the PD‑L1 gene and protein in MG‑63 cell was greater than HOS and U2OS cells. The specific lysis of human osteosarcoma cells induced by NK cells was enhanced when PD‑L1/PD‑1 was blocked by the PD‑L1 antibody. The present study proposed that the PD‑L1/PD‑1 axis serves an important role in NK cell‑induced cytotoxicity in osteosarcoma via granzyme B secretion. Our findings may contribute to the development of precise treatments for osteosarcoma based on the expression profile of PD‑L1 in patients with this disease.