This study investigates the role of HDAC6 in α-tubulin lactylation, a posttranslational modification that regulates microtubule dynamics. The authors identify lysine 40 (K40) of α-tubulin as the site of lactylation, which is catalyzed by HDAC6 using lactate as a donor. Lactylation of α-tubulin enhances microtubule dynamics and promotes neurite outgrowth and branching in cultured hippocampal neurons. The study reveals that HDAC6 acts as the primary lactyltransferase, with its deacetylase activity being crucial for lactylation. The lactylation process is reversible and dependent on lactate concentrations. Additionally, the authors demonstrate that glycolysis, particularly through lactate production, regulates α-tubulin lactylation. They also show that Sirt2, another deacetylase, removes lactylated α-tubulin. The conservation of lactylation activity among HDAC family proteins is discussed, suggesting a potential role in regulating cytoskeleton functions. Overall, the findings establish a link between cell metabolism and cytoskeleton dynamics through α-tubulin lactylation.This study investigates the role of HDAC6 in α-tubulin lactylation, a posttranslational modification that regulates microtubule dynamics. The authors identify lysine 40 (K40) of α-tubulin as the site of lactylation, which is catalyzed by HDAC6 using lactate as a donor. Lactylation of α-tubulin enhances microtubule dynamics and promotes neurite outgrowth and branching in cultured hippocampal neurons. The study reveals that HDAC6 acts as the primary lactyltransferase, with its deacetylase activity being crucial for lactylation. The lactylation process is reversible and dependent on lactate concentrations. Additionally, the authors demonstrate that glycolysis, particularly through lactate production, regulates α-tubulin lactylation. They also show that Sirt2, another deacetylase, removes lactylated α-tubulin. The conservation of lactylation activity among HDAC family proteins is discussed, suggesting a potential role in regulating cytoskeleton functions. Overall, the findings establish a link between cell metabolism and cytoskeleton dynamics through α-tubulin lactylation.