Inflammatory tolerance is a crucial mechanism that limits inflammatory responses in order to avoid prolonged inflammation that may damage the host. Evidence that chronic inflammation contributes to the neuropathology of prevalent neurodegenerative and psychiatric diseases suggests that inflammatory tolerance mechanisms are often inadequate to control detrimental inflammation in the central nervous system. Thus, identifying mechanisms that regulate neuroinflammatory tolerance may reveal opportunities for bolstering tolerance to reduce chronic inflammation in these diseases. Examination of tolerance after repeated lipopolysaccharide (LPS) treatment of mouse primary astrocytes demonstrated that histone deacetylase (HDAC) activity promoted tolerance, opposite to the action of glycogen synthase kinase-3 (GSK3), which counteracts tolerance. HDAC6 in particular was found to be critical for tolerance induction, as its deacetylation of acetyl-tubulin was increased during LPS tolerance, this was enhanced by inhibition of GSK3, and the HDAC6 inhibitor tubacin completely blocked tolerance and the promotion of tolerance by inhibition of GSK3. These results reveal opposing interactions between HDAC6 and GSK3 in regulating tolerance, and indicate that shifting the balance between these two opposing forces on inflammatory tolerance can obliterate or enhance tolerance to LPS in astrocytes.