Endothelial cell damage, such as apoptosis and necrosis, is involved in many cardiovascular diseases. In recent years, the crucial role of microRNAs in controlling tissue homeostasis and disease in the epithelium has become widely recognized. In the present study, human umbilical vein endothelial cells were transfected with a miRNA agomir and a SMAD4 expression vector. The expression of miR125b-5p was determined by using quantitative real-time polymerase chain reaction. Cell apoptosis and necrosis were measured with flow cytometry. The expression of SMAD4 was evaluated with Western blotting. Here, we demonstrated that the rates of apoptosis and necrosis were significantly decreased in the miR125b-5p agomir group of HUVECs under H₂O₂-induced oxidative stress compared with the miR125b-5p antagomir group. Further experiments revealed that the expression of SMAD4 is negatively regulated by miR125b-5p. Moreover, we identified that the rates of apoptosis and necrosis were increased when SMAD4 and miR125b-5p were both overexpressed compared with miR125b-5p overexpression alone. The present study demonstrates for the first time that the overexpression of miR125b-5p can reduce H₂O₂-induced oxidative damage via SMAD4, suggesting that miR125b-5p has therapeutic potential for preventing oxidative stress-related diseases.