Drug resistance remains a major obstacle in neuroblastoma treatment. Lysosomal sequestration, facilitated by the V-ATPase proton pump, is one of the mechanisms of chemoresistance. Overexpression of the ATP6V0D1 subunit of V-ATPase, previously reported in various cancers, was also observed in ellipticine-resistant neuroblastoma cells in our study. Neuroblastoma cells also exhibited increased lysosomal capacity and vacuolation after ellipticine treatment. Knockdown of ATP6V0D1, but not ATP6V1H, enhanced ellipticine sensitivity, suppressed proliferation and migration, decreased lysosomal uptake, and induced G2/M arrest in neuroblastoma cell lines. Notably, inhibiting another V-ATPase subunit, ATP6V1H, had no effect, highlighting the specific role of ATP6V0D1 in drug resistance. Ellipticine-induced vacuolation, identified as endoplasmic reticulum swelling, lacked evidence of paraptosis. ATP6V0D1 knockdown suppressed this phenomenon, whereas ATP6V1H silencing did not. Our findings underscore the importance of ATP6V0D1 in neuroblastoma and suggest potential therapeutic strategies targeting V-ATPase for overcoming drug resistance.