Withaferin A (WFA) is a steroidal lactone derived from Withania somnifera, a plant cornerstone in Ayurvedic medicine for over 3000 years. WFA exhibits antitumoral properties against glioblastoma (GBM), but its mechanisms remain incompletely understood. We investigated its effect on the intermediate filament nestin, a marker of GBM stemness and poor prognosis. WFA treatment induced a partial, reversible collapse of nestin networks, characterized by juxtanuclear accumulation. WFA also decreased the elasticity of GBM cells, inhibited their proliferation and migration, increased their sensitivity to serum deprivation and produced profound mitochondrial alterations, strongly resembling the phenotype caused by genetic deletion of the sacsin chaperone in these cells. Such correlation could be explained by the fact that WFA downregulated sacsin at both mRNA and protein levels. RNA seq analysis indicated that the incubation with WFA and sacsin loss produced distinct expression profiles, but shared alterations in cell survival and response to stress pathways. Most importantly, WFA inhibited the expression of major transcriptional regulators associated to GBM malignancy with binding motifs in the rat, mouse and human promoters of sacsin. These results suggest a novel mechanism whereby WFA could exert its antitumoral effects sacsin downregulation, destabilization of nestin filaments and impaired resistance to stress. Furthermore, our results support a role for sacsin as an oncogene and putative therapeutic target in GBM, as recently suggested for other types of cancer.