Valproic acid (VPA), used to treat bipolar mood disorder and seizures, also inhibits histone deacetylase (HDAC). Here, we found that VPA and other HDAC inhibitors, butyrate and trichostatin A, robustly protected mature cerebellar granule cell cultures from excitotoxicity induced by SYM 2081 ((2S, 4R)-4-methylglutamate), an inhibitor of excitatory amino-acid transporters and an agonist of low-affinity kainate receptors. These neuroprotective effects required protracted treatment and were correlated with enhanced acetylated histone levels, indicating HDAC inhibition. SYM-induced excitotoxicity was blocked by MK-801 ((5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate), supporting that the toxicity was largely N-methyl-D-aspartate receptor dependent. SYM excitotoxicity had apoptotic characteristics and was prevented by a caspase inhibitor. SYM-induced apoptosis was associated with a rapid and robust nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene previously shown to be proapoptotic. VPA pretreatment suppressed SYM 2081-induced GAPDH nuclear accumulation, concurrent with its neuroprotective effects. Chromatin immunoprecipitation (ChIP) revealed that GAPDH is copresent with acetylated histone H3, including Lys9-acetylated histone, and that VPA treatment caused a time-dependent decrease in the levels of nuclear GAPDH with a concomitant increase in acetylated histones in the ChIP complex. Our results strongly suggest that VPA protects neurons from excitotoxicity through inhibition of HDAC activity and that this protective effect may involve suppression of excitotoxicity-induced accumulation of GAPDH protein in the nucleus.