Neuroblastoma is an aggressive childhood disease of the sympathetic nervous system. Treatments are often ineffective and have serious side effects. Conventional therapy of neuroblastoma includes the differentiation agents. Unlike chemo-radiotherapy, differentiation therapy shows minimal side effects on normal cells, because normal non-malignant cells are already differentiated. Keeping in view the limited toxicity of Withania somnifera (Ashwagandha), the current study was aimed to investigate the efficacy of Ashwagandha water extract (ASH-WEX) for anti-proliferative potential in neuroblastoma and its underlying signalling mechanisms. ASH-WEX significantly reduced cell proliferation and induced cell differentiation as indicated by morphological changes and NF200 expression in human IMR-32 neuroblastoma cells. The induction of differentiation was accompanied by HSP70 and mortalin induction as well as pancytoplasmic translocation of the mortalin in ASH-WEX treated cells. Furthermore, the ASH-WEX treatment lead to induction of neural cell adhesion molecule (NCAM) expression and reduction in its polysialylation, thus elucidating its anti-migratory potential, which was also supported by downregulation of MMP 2 and 9 activity. ASH-WEX treatment led to cell cycle arrest at G0/G1 phase and increase in early apoptotic population. Modulation of cell cycle marker Cyclin D1, anti-apoptotic marker bcl-xl and Akt-P provide evidence that ASH-WEX may prove to be a promising phytotherapeutic intervention in neuroblatoma related malignancies.