Hyperglycemia is one of the major factors responsible for the myocardial apoptosis and dysfunction in diabetes. Many studies have proved that there is a close relationship between decreased Na+/K+-ATPase activity and diabetic cardiomyopathy. However, the effect of directly activated Na+/K+-ATPase on high glucose-induced myocardial injury is still unknown. Here we found that DRm217, a Na+/K+-ATPase's DR-region specific monoclonal antibody and direct activator, could prevent high glucose-induced H9c2 cell injury, reactive oxygen species (ROS) release, and mitochondrial dysfunction. High glucose-treatment decreased Na+/K+-ATPase activity and increased intracellular Ca2+ level, whereas DRm217 increased Na+/K+-ATPase activity and alleviated Ca2+ overload. Inhibition of Ca2+ overload or closing sodium calcium exchanger (NCX channel) could reverse high glucose-induced ROS increasing and cell injury. In addition, DRm217 could significantly attenuate high glucose-induced p38, JNK and ERK1/2 phosphorylation, which were involved in high glucose-induced cell injury and ROS accumulation. Our findings suggest that DRm217 may protect against the deleterious effects of high glucose in the heart. Prevention of high glucose-induced myocardial cell injury by specific Na+/K+-ATPase activator may be an attractive therapeutic option.