HDAC inhibitors have been reported as potential therapeutic agents for the neurodegenerative diseases such as Alzheimer disease and Parkinson disease. In this study, we examined which molecules HDAC inhibitors regulate for the therapeutic effects in primary neuronal cells. Western blot analysis revealed that HDAC inhibitors such as Valproic acid, Trichostatin A and FK228 decreased protein expression of p35 and p35 cleavage into p25 in response to ionomycin, glutamate and amyloid beta. The treatment of proteasome inhibitors reversed HDAC inhibitor-decreased expression of p35 protein, but HDAC inhibitor still decreased proteasome inhibitor-increased p25 protein level, suggesting that proteasome activation mediates HDAC inhibitor-decreased p35 protein level. Furthermore, HDAC inhibitors increased mRNA expression of calpastatin in primary neuronal cells. The transfection of calpastatin siRNA reversed HDAC inhibitor-decreased p25 protein level in SH-SY5Y cells. Our results demonstrate that HDAC inhibitors regulate the protein expression and cleavage of p35 through proteasome activation and calpastatin expression, respectively.