Endothelial to mesenchymal transition (EndMT) serves as a crucial source of myofibroblasts and exerts a significant impact on fibrosis diseases through an unidentified underlying mechanism. Sirtuin6 (Sirt6) is widely involved in the physiology and pathology of cardiovascular diseases. However, the role of Sirt6 on the EndMT in viral myocarditis (VMC) remains unclear. This study aimed to investigate the effect of Sirt6 on the EndMT in coxsackievirus B3 (CVB3)-infected mouse cardiac endothelial cells (MCECs). Firstly, the occurrence of EndMT was confirmed in CVB3-induced MCECs, and a down-regulation of Sirt6 expression was observed during this process. To further explore the impact of Sirt6 on EndMT, the expression of Sirt6 in MCECs was manipulated using lentiviral transfection. The results showed that Sirt6 over-expression led to a up-regulation in the endothelial marker ve-Cadherin and a concomitant down-regulation in the mesenchymal marker α-Sma, partially reversing the EndMT caused by CVB3 stimulation or Sirt6 deficiency. Flow cytometry and western blot were used to detect apoptosis, and the results indicated that Sirt6 over-expression might reduce apoptosis through the Caspase-3 pathway. Subsequently, proteomic analysis was conducted based on MCECs with Sirt6 knockdown. The results suggested that Sirt6 deficiency was closely associated with apoptosis, biological oxidation, and energy metabolism. Further detection of oxidative stress and autophagy levels in MCECs revealed that CVB3 stimulation led to enhanced oxidative stress and reduced autophagy. This condition was exacerbated by lack of Sirt6 but partially mitigated by its over-expression. These findings suggested that Sirt6 might inhibit the EndMT by ameliorating oxidative stress and autophagy in CVB3-induced MCECs.