A number of agents reducing interleukin-1β (IL-1β) activity are being developed as novel immunomodulatory and anti-inflammatory therapies. However, the elucidation of their molecular mechanism of action is required in the context of medical management of inflammatory diseases. Inhibitors of histone deacetylases (HDACs) are promising anticancer agents with pleiotropic activities. Of these, suberoylanilide hydroxamic acid has been reported to inhibit the production of several proinflammatory cytokines. In the present study, we investigated the effects of 2 HDAC inhibitors on IL-1β secretion: suberoylanilide hydroxamic acid and a newly developed hydroxamic acid-derived compound ITF2357. These HDAC inhibitors do not affect the synthesis or intracellular localization of IL-1β but both strongly reduce the levels of extracellular IL-1β by preventing the exocytosis of IL-1β-containing secretory lysosomes. At nanomolar concentrations, ITF2357 reduces the secretion of IL-1β following ATP activation of the P2X7 receptor. Whereas the inhibition of HDACs results in hyperacetylation of tubulin, acetylation of HSP90 was unaffected. The reduction in IL-1β secretion appears to be due to disruption of microtubules impairing lysosome exocytosis. Together, these observations indicate that a functional microtubule network is required for IL-1β secretion and suggest that disruption of tubulin is the mechanism by which inhibitors of HDACs reduce the secretion of IL-1β.