Among pediatric solid tumors, brain tumors are the ones that cause more mortality. Although the survival rates for some pediatric brain tumors have improved in recent years, the overall prognosis remains poor. In addition, the long-term survivors suffer severe cognitive and psychological sequels due to the treatments used. Therefore, there is still an urgent need to develop new therapies that not only cure the patients but also are less toxic for them. Oncolytic viruses have emerged as a promising therapeutic tool due to their ability to replicate selectively in tumor cells, keeping healthy cells intact, as well as to induce systemic antitumor immunity. VCN-01 is a replication-competent oncolytic adenovirus whose efficacy for treating several brain tumors, such as high-grade gliomas, has already been tested in preclinical models. However, before clinical trials can be performed, it must be verified that its intracranial administration is safe. This study analysed the VCN-01 toxicity and biodistribution after an intracranial injection in a Syrian hamster model. The dosages that were employed were 1.5x10⁹ vp/animal and 1.5x10¹⁰ vp/animal, which were equivalent to 5 and 50 fold higher than the starting clinical dose (1x10¹⁰ vp/patient). Our toxicity results showed that VCN-01 intracranial administration has a safe profile with no associated adverse effects. Regarding biodistribution, VCN-01 remained confined mainly to the brain, being its presence in other tissues residual. In conclusion, the toxicity and biodistribution results obtained in this study support the safety profile of an intracranial administration of VCN-01 for the treatment of patients with brain tumors.