Downstream products of alcohol metabolism can impact the level of acetylated alpha-tubulin through the production of acetyl-CoA and the effects of acetylase alpha-tubulin N acetyltransferase1 (ATAT1) and deacetylase histone deacetylase6 (HDAC6) expressions. To determine the impact of ethanol intake on the modulation of tubulin acetylation in the brain, we administered ethanol to mice for different durations and examined their brain tissues. We used wild-type and HDAC6 null adult male and female mice. The control groups received PBS, and the treatment groups received ethanol (20% in PBS, 2g/kg) intraperitoneally. We analyzed the acetyl-CoA and CoA metabolites using high-performance liquid-chromatography (HPLC) methods. Furthermore, we determined changes in the acetylated alpha-tubulin levels and the enzymes regulating alpha-tubulin acetylation by western blots. We observed sex-related changes in the ethanol-induced hyperacetylation of alpha-tubulin in mice cerebellum and hippocampus. These differences can be attributed to the changes in the ethanol-induced expression levels of the acetylase/deacetylase enzymes.