Histone deacetylases are an important class of enzymes that catalyze the hydrolysis of acetyl-L-lysine side chains in histone and non-histone proteins to yield L-lysine and acetate, effecting the epigenetic regulation of gene expression. In addition to the active site pocket, the enzyme harbors an internal cavity for the release of acetate by-product. To probe the role of highly conserved amino acid residues lining this exit tunnel, site-directed alanine substitutions were made at tyrosine-18, tyrosine-20 and histidine-42 positions. These mutants were characterized by various biochemical and biophysical techniques to define the effect of mutations on ligand binding and catalysis of the enzyme. The mutations altered the catalytic activity of HDAC8 significantly. Y18A mutation dramatically impaired the structural-functional aspects of the enzymatic reaction. Our data reveal that there is long range communication between the exit tunnel residues and the active site pocket of HDAC8, presumably regulating the overall catalysis of the enzyme.