Virotherapy using oncolytic viruses is an upcoming therapy strategy for cancer treatment. A variety of preclinical and clinical trials have indicated that adenoviruses may be used as potent agents in the treatment of a variety of cancers, and also for the treatment of brain tumors. In these studies, it has also been shown that oncovirotherapy is safe in terms of toxicity and side effects. In addition, previous studies have presented evidence for a significant role of oncovirotherapy in the activation of anti‑tumor immune responses. With regard to oncolytic adenoviruses, we have demonstrated previously that the multifunctional protein Y‑box binding protein‑1 (YB‑1) is a potent factor that was used to develop an YB‑1‑dependent oncolytic adenovirus (XVir‑N‑31). XVir‑N‑31 provides the opportunity for tumor‑selective replication and exhibited marked oncolytic properties in a mouse glioma tumor model using therapy‑resistant brain tumor initiating cells (BTICs). In a number of, but not all, patients with glioma, YB‑1 is primarily located in the nucleus; this promotes XVir‑N‑31‑replication and subsequently tumor cell lysis. However, in certain BTICs, only a small amount of YB‑1 has been identified to be nuclear, and therefore virus replication is suboptimal. YB‑1 in BTICs was demonstrated to be translocated into the nucleus following irradiation, which was accompanied by an enhancement in XVir‑N‑31 production. R28 glioma spheres implanted in living organotypic human brain slices exhibited a significantly delayed growth rate when pre‑irradiated prior to XVir‑N‑31‑infection as compared with single treatment methods. Consistent with the in vitro data, R28 glioma‑bearing mice exhibited a prolonged mean and median survival following single tumor irradiation prior to intratumoral XVir‑N‑31 injection, compared with the single treatment methods. In conclusion, the present study demonstrated that in an experimental glioma model, tumor irradiation strengthened the effect of an XVir‑N‑31‑based oncovirotherapy.