Background: Diabetes has been found to induce various subtle molecular changes important for sperm quality and function: The mammalian spermatozoa are rich in polyunsaturated fatty acids, making them particularly sensitive to the deleterious effects of lipid peroxidation, which may result in irreversible loss of motility and a low level of fertilizing capacity. An antioxidant, glutathione (GSH) a polypeptide is important in biological oxidation-reduction reactions and improves sperm motility. Objective: The study was aimed to investigate the effects of GSH injection on healthy and diabetic male mice fertilization potential in vitro following in vitro fertilization (IVF). Materials and Methods: Inducing diabetes in mice through intra peritoneal alloxan injection. The treatment with GSH divided into two periods 10 and 20 days, studying the in vitro fertilization outcomes. Results: Glutathione daily IV injected in a period of 10 to 20 days for the diabetic induced male mice significantly increase the sperm concentration and motility and decrease the abnormal and dead sperms percentages in treated group compared to untreated group. The Fertilization rate was significantly (P<0.05) increased in mice treated with GSH compared to untreated one. Conclusion: Glutathione injections may decrease the oxidative stress caused by diabetes and enhance the in vitro fertilization potentials in male mice.