The potential mechanism of high glucose-induced cardiomyocyte apoptosis and selenium's protective effects were investigated in this study. Myocytes isolated from neonate rats were cultured in high-glucose medium (25.5 mmol/L glucose) to mimic sustained hyperglycemia. Before high-glucose incubation, myocytes were pretreated by sodium selenite solution. Cell apoptosis was evaluated by annexin V/propidium iodide (PI) staining and caspase activation. Expression of Toll-like receptor 4 (TLR-4) and myeloid differentiation factor 88 (MyD-88) was examined at both mRNA and protein levels. The intracellular reactive oxygen species (ROS) production and glutathione peroxidase (GPx) activity in myocytes were also detected. We found high glucose-induced cell apoptosis and activation of TLR-4/MyD-88/caspase-8/caspase-3 signaling, accompanied by increased production of ROS. Selenium pretreatment attenuated apoptosis in high glucose-incubated myocytes, and mechanically, this protective effect was found to be associated with attenuating oxidative status by increasing activity of GPx, decreasing the generation of ROS, as well as inhibition of the activation of TLR-4/MyD-88/caspase-8/caspase-3 signaling in myocytes. These results suggest that activation of TLR-4/MyD-88 signaling pathway plays an important role in high glucose-induced cardiomyocyte apoptosis. Additionally, by modulating TLR-4/MyD-88 signaling pathway, which is linked to ROS formation, selenium exerts its antioxidative and antiapoptotic effects in high glucose-incubated myocytes.