Neuroblastoma (NB) is among the most aggressive pediatric solid tumors, and outcomes for patients with high-risk or relapsed disease remain poor despite intensive multimodal therapy. Oncolytic adenoviruses represent a promising therapeutic approach; however, achieving tumor-selective replication while maintaining robust antitumor efficacy remains a major challenge. Paired-like homeobox 2B (PHOX2B) is a transcription factor that is broadly expressed in NB and largely absent from normal postnatal tissues, making its promoter an attractive candidate for transcriptional targeting. In this study, we developed a PHOX2B promoter-driven oncolytic adenovirus, Ad-pPHOX2B-5F, in which adenoviral E1 expression is controlled by the PHOX2B promoter. Using NB and non-NB cell lines, as well as isogenic models with PHOX2B overexpression, we examined the relationship between PHOX2B expression, viral gene activation, viral genome amplification, and cytotoxicity, and we evaluated antitumor efficacy in NB xenograft models. Ad-pPHOX2B-5F induced potent, selective cytotoxicity in PHOX2B -expressing NB cells, whereas cells with low or absent PHOX2B expression were largely resistant. Viral genome amplification and cytotoxicity were associated with PHOX2B expression, and PHOX2B overexpression in resistant G401 cells was accompanied by increased PHOX2B promoter activity, viral genome amplification, and susceptibility to Ad-pPHOX2B-5F. Notably, in selected NB cell lines, rapid cytotoxicity was observed despite limited detectable viral genome amplification and was accompanied by bystander cytotoxicity associated with filtered supernatants. In vivo , intratumoral administration of Ad-pPHOX2B-5F suppressed tumor growth in PHOX2B-positive xenografts and was associated with a reduction in PHOX2B-positive tumor areas. Together, these findings support the therapeutic potential of PHOX2B promoter-driven oncolytic adenoviruses for high-risk NB.