This study reveals that HDAC6 catalyzes α-tubulin lactylation, a post-translational modification that regulates microtubule dynamics and cytoskeleton functions. α-Tubulin lactylation occurs on lysine 40 (K40) and is enhanced by high lactate concentrations. HDAC6, a deacetylase, acts as the primary lactyltransferase for α-tubulin lactylation, and this process is reversible and dependent on lactate levels. The study shows that lactylation of α-tubulin increases microtubule dynamics, promotes neurite outgrowth and branching in cultured neurons, and enhances axonal regeneration after injury. Additionally, HDAC6's lactyltransferase activity is conserved among HDAC family proteins, with HDAC3 also playing a role in lactylating microtubule-associated proteins. The findings establish a link between cell metabolism and cytoskeleton functions, highlighting the importance of lactylation in regulating microtubule dynamics and neuronal development. The study also demonstrates that lactylation and acetylation compete for the same residue on α-tubulin, with lactylation promoting microtubule dynamics while acetylation marks stable microtubules. The results suggest that α-tubulin lactylation may play a critical role in neuronal morphogenesis and axonal transport, and further research is needed to understand its physiological and pathological functions in vivo.This study reveals that HDAC6 catalyzes α-tubulin lactylation, a post-translational modification that regulates microtubule dynamics and cytoskeleton functions. α-Tubulin lactylation occurs on lysine 40 (K40) and is enhanced by high lactate concentrations. HDAC6, a deacetylase, acts as the primary lactyltransferase for α-tubulin lactylation, and this process is reversible and dependent on lactate levels. The study shows that lactylation of α-tubulin increases microtubule dynamics, promotes neurite outgrowth and branching in cultured neurons, and enhances axonal regeneration after injury. Additionally, HDAC6's lactyltransferase activity is conserved among HDAC family proteins, with HDAC3 also playing a role in lactylating microtubule-associated proteins. The findings establish a link between cell metabolism and cytoskeleton functions, highlighting the importance of lactylation in regulating microtubule dynamics and neuronal development. The study also demonstrates that lactylation and acetylation compete for the same residue on α-tubulin, with lactylation promoting microtubule dynamics while acetylation marks stable microtubules. The results suggest that α-tubulin lactylation may play a critical role in neuronal morphogenesis and axonal transport, and further research is needed to understand its physiological and pathological functions in vivo.