Diabetic cardiomyopathy (DCM), a cardiac condition resulting from diabetes, is linked to significant morbidity and mortality rates. TUBB4A, a variant of tubulin, is highly expressed in heart-related illnesses, yet its function in DCM remains unclear. In this study, 60 mg/kg streptozotocin (STZ) was intraperitoneally injected into mice to induce a diabetic model, and 30 mM glucose was added to H9C2 cell medium for 48 h to induce a high glucose (HG) cell model. In this study, TUBB4A expression was decreased in STZ- or HG-induced animal or cellular DCM models. The overexpression of TUBB4A diminished the effects of STZ or HG, enhanced the growth of myocardial cells, and prevented their hypertrophy and apoptosis. Moreover, it inhibited the expression of ROS, Bax and C-Caspase-3
promoted the expression of Bcl-2 and also alleviated DCM in vivo. Mechanistically, TUBB4A interacts with MYH9 and promotes NOTCH1 expression through MYH9-mediated deubiquitination, thereby inhibiting HG-induced cardiomyocyte hypertrophy and apoptosis and alleviating the development of DCM. Our study suggests that increasing TUBB4A expression may be a potential strategy for the treatment of DCM.