Selenium is a nutrient that, applied in appropriate concentrations, has many positive effects on plants. However, Selenium excessive concentration treatment leads to pro-oxidation reactions, which results in manifestations of toxicity and oxidative stress. Glutathione, as chelating agent, antioxidant, reducing agent and the signal component, has a central role in the metabolism of Selenium and antioxidant defense of plants. With the aim of investigating the potential role of glutathione in maintaining a balance in the absorption of Selenium and the prevention of stress, the seeds of wheat (Triticum aestivum) were treated with three different concentrations of two different forms of selenium (selenate and selenite solutions). There was a significant increase in the concentration of total glutathione content (tGSH, GSH+GSSG) in wheat shoots, but only in the case of application of the highest concentrations of selenite (1 mg/kg) and there was a significant increase in activities of glutathione peroxidase (GPx). Therefore, a positive correlation was found between the concentration of tGSH and antioxidant activity of GPx's. Obtained results suggest the involvement of GSH and GPx subjected to selenium supplementation in wheat, therefore making an effective defense system of plants by participating in antioxidant reactions, which allow the regulation of ROS formation and prevent damage of plant tissue as a result of oxidative stress.