Withaferin A (WIT) is a natural product possessing a wide range of pharmacologic activities. Previous studies have reported covalent binding of WIT to tubulin and down-of tubulin protein levels although the underlying mechanisms remain to be established. In the current investigation, we showed that WIT induces down-regulation of tubulin in a post-transcriptional manner, suggestive of direct and potent activity in tubulin degradation. The ,'-ethylene bis(iodoacetamide) assay and competitive binding experiments with four colchicine site-targeted tubulin inhibitors further revealed that WIT interacts with the colchicine site of tubulin to promote degradation. WIT irreversibly inhibited tubulin polymerization, and mass spectrometry results disclosed binding to cysteine at position 239 (Cys239) and Cys303 sites of -tubulin. Interestingly, WIT promoted degradation of the -tubulin isoforms containing Cys239 [2, 4, and 5()] but had no effect on those containing Ser239 (3 and 6). Moreover, a C239S but not C303S mutation in -tubulin completely abolished the degradation effect of WIT, suggesting that the Cys239-WIT covalent bond accounts for this activity. Our collective results clearly demonstrate that covalent interactions between WIT and Cys239 of -tubulin promote tubulin degradation, supporting its potential utility as a therapeutic compound. SIGNIFICANCE STATEMENT: Withaferin A, a natural product possessing a wide range of pharmacologic activities, covalently binds to Cys239 of -tubulin near the colchicine site, and the WIT-Cys239 covalent bond accounts for WIT-induced tubulin degradation, fully clarifying the underlying mechanisms and supporting its potential utility a therapeutic compound.