Metformin has been shown to ameliorate diabetic cardiomyopathy. In the present research we investigatedwhether metformin would reduce cardiomyocyte apoptosis that was induced by high-glucose stimulation in vitrovia activation of PP2A. Primary human and rat cardiomyocytes were subject to high-glucose stimulation. Okadaicacid was used to inhibit PP2A activity. Cell viability and apoptosis was assessed using CCK-8 and by flowcytometry, respectively. Release of HMGB1, TNF-alpha or IL-6 was analyzed by ELISA. Oxidative stress wasevaluated by measuring cellular ROS and mitochondrial superoxide level. PP2A activity was evaluated by Serine/Threonine phosphatase assay system or analyzing Y307 phosphorylation level of PP2A catalytic domain (PP2Ac)by Western blot and the association between PP2Ac and alpha-4 by co-immunoprecipitation. Activation of the NF-kappa-Bsignaling pathway was assessed by detecting Ser32 phosphorylation level of I-kappa-Ba as well as nuclear entry of p65protein by Western blot. Activation of the GSK3-beta/MCL1 signaling pathway was assessed by detecting Ser9phosphorylation level of GSK3-beta and protein level of MCL1. We found Metformin pre-treatment attenuatedhuman and rat cardiomyocytes apoptosis,HMGB1, TNF-alpha and IL-6 release and ROS production that were inducedby high-glucose stimulation, and these effects of metformin could be blocked by okadaic acid treatment. Metforminreduced the upregulation of PP2Ac pY307 and the PP2Ac-alpha-4 association, which was not affected byokadaic acid treatment. Metformin pre-treatment reduced NF-kappa-B activation in human and rat cardiomyocytesapoptosis that was elicited by high-glucose stimulation, and this effect of metformin could be blocked by okadaicacid treatment. GSK3-beta/MCL1 is not part of metformin activating PP2A induced myocardial cell death inhibition.In conclusion, metformin reduced apoptosis, ROS production and inflammatory response in primary human andrat cardiomyocytes in vitro in a PP2A dependent manner.