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＜sup＞+＜/sup＞/K＜sup＞+＜/sup＞-ATPase　activity　and　diabetic　cardiomyopathy.　However,　the　effect　of　directly　activated　Na＜sup＞+＜/sup＞/K＜sup＞+＜/sup＞-ATPase　on　high　glucose-induced　myocardial　injury　is　still　unknown.　Here　we　found　that　DRm217,　a　Na＜sup＞+＜/sup＞/K＜sup＞+＜/sup＞-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＜sup＞+＜/sup＞/K＜sup＞+＜/sup＞-ATPase　activity　and　increased　intracellular　Ca＜sup＞2+＜/sup＞　level,　whereas　DRm217　increased　Na＜sup＞+＜/sup＞/K＜sup＞+＜/sup＞-ATPase　activity　and　alleviated　Ca＜sup＞2+＜/sup＞　overload.　Inhibition　of　Ca＜sup＞2+＜/sup＞　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＜sup＞+＜/sup＞/K＜sup＞+＜/sup＞-ATPase　activator　may　be　an　attractive　therapeutic　option.