AbstractSkin fibrosis, a pathological process featured by fibroblast activation and extracellular matrix (ECM) deposition, makes a significant contribution to morbidity. Studies have identified biomechanics as the central element in the complex network of fibrogenesis that drives the profibrotic feedback loop. In this study, we found that the acetylation of α‐tubulin at lysine 40 (K40) was augmented in fibrotic skin tissues. Further analysis showed that α‐tubulin acetylation is required for fibroblast activation, including contraction, migration, and ECM deposition. More importantly, we revealed that biomechanics‐induced upregulation of K40 acetylation promotes fibrosis by mediating mechanosensitive Yes‐associated protein S127 dephosphorylation and its cytoplasm nucleus shuttle. Furthermore, we demonstrated that the knockdown of α‐tubulin acetyltransferase 1 could rescue the K40 acetylation upregulation caused by increased matrix rigidity and ameliorate skin fibrosis both in vivo and in vitro. Herein, we highlight the critical role of α‐tubulin acetylation in matrix stiffness‐induced skin fibrosis and clarify a possible molecular mechanism. Our research suggests α‐tubulin acetylation as a potential target for drug design and therapeutic intervention.