Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. One fundamental issue regarding NB recurrence and metastasis is the maintenance of cancer stem cells (CSCs) stemness. Tankyrase 1 (TNKS1) is overexpressed in several types of cancers and in NB cell lines. XAV939 is a small molecule inhibitor of TNKS1 and can induce apoptosis of NB cells. In this study, we showed that the surface marker CD133 method was more suitable for isolating NB CSCs than the side-population method, and 60 µM etoposide was optimal for enriching NB CSCs. The NB CSCs were demonstrated in juvenescence or stemness state by electron microscopy, which was in line with the characteristics of CSCs. Furthermore, we demonstrated that the expression of the CSCs marker CD133 and migration ability of CSCs decreased after XAV939 treatment or by RNAi‑mediated knockdown of the TNKS1 gene. These findings suggest that XAV939 treatment or RNAi-TNKS1 inhibits the stemness and migration of NB CSCs via the repression of TNKS1, and TNKS1 may be a potential molecular target for eliminating NB CSCs by small molecule drugs.