The post-translational modifications (PTMs) occurring on microtubules have been implicated in the regulation of microtubule properties and functions. Acetylated K40 of α-tubulin, a hallmark of long-lived stable microtubules, is known to be negatively controlled by histone deacetylase 6 (HDAC6). However, the vital roles of HDAC6 in microtubule-related processes such as cell motility and cell division cannot be fully explained by the only known target site on tubulin. Here, we attempt to comprehensively map lysine acetylation sites on tubulin purified from mouse brain tissues. Furthermore, mass spectrometry-based quantitative comparison of acetylated peptides from wild-type vs HDAC6 knockout mice allowed us to identify six new deacetylation sites possibly mediated by HDAC6. Thus, adding new sites to the repertoire of HDAC6-mediated tubulin deacetylation events would further our understanding of the multi-faceted roles of HDAC6 in regulating microtubule stability and cellular functions.