Glioma is resistant to the apoptotic effects of chemotherapy and the mechanism underlying its chemoresistance is not currently understood. In a previous study, we reported that osteopontin (OPN) was overexpressed in glioma tissues and had an important anti‑apoptotic effect. Furthermore, overexpression of OPN was observed following chemotherapy. To elucidate whether OPN plays a role in chemotherapy resistance and to investigate its downstream signaling pathway, this study used small interfering RNA (siRNA) to silence the expression of OPN in U251 human neuronal glioma astrocytoma cells. OPN downregulation in U251 cells enhanced the apoptotic effects induced by temozolomide (TMZ) and cisplatin (DDP). Furthermore, OPN siRNA suppressed the nuclear factor κ‑light‑chain‑enhancer of activated B cells (NF‑κB) activation and B cell lymphoma 2 (Bcl‑2) expression that was induced by chemotherapy. Taken together, these results demonstrated that the expression levels of OPN are involved in glioma chemoresistance. Knockdown of OPN through siRNA enhanced the effects of TMZ and DDP chemotherapy by targeting the NF‑κB/Bcl‑2 pathway.