Neuroblastoma (NB) is the most common extracranial tumor of childhood with poor prognosis in a high-risk group. An obstacle in the development of treatment for solid tumors is the immunosuppressive nature of the tumor microenvironment (TME). Regulatory T cells (Tregs) represent a T cell subset with specialized function in immune suppression and maintaining self-tolerance. Tregs resident within the tumor milieu is believed to play an important role in immune escape mechanisms. The role of the NB microenvironment in promoting Treg phenotype has never been elucidated. Herein, we demonstrated that the NB microenvironment promoted T cell activation and one NB cell line, SK-N-SH, manifested an ability to induce Treg differentiation. We identified tumor-derived HMGB1 as a potential protein responsible for Treg phenotype induction. By neutralizing HMGB1, Treg differentiation was abolished. Finally, we adopted a dataset of 498 pediatric NB via the NCBI GEO database, accession GSE49711, to validate clinical relevance of HMGB1 overexpression. Up to 11% of patients had HMGB1-overexpressed tumors. Moreover, this patient subpopulation showed higher risks of tumor progression, relapse, or death. Our findings emphasize the importance of immunological signature of tumor cells for appropriate therapeutic approach. Upregulation of secretory HMGB1 may contribute to suppression of antitumor immunity through induction of Tregs in the NB microenvironment.