Objective AG129 mice have difficulty giving birth naturally, and conventional in vitro fertilization (IVF) reagents do not yield a sufficient number of embryos for transplantation. Reduced glutathione (GSH) was used to regulate a component of the IVF reagent and complete the IVF process in AG129 mice, presenting a potential method for biological purification and rapid reproduction of genetically engineered mouse strains with IVF difficulties. Methods Four experimental groups were designed using 0 mmol / L (control group), 0. 5 mmol / L, 1 mmol / L and 2 mmol / L of GSH for sperm capacitation and fertilization. Each group contained 5 superovulated mice, and the assay was repeated three times. Results The average fertilization rate differed between the GSH experimental groups and the control group (P< 0. 05). The 1 mmol / L GSH concentration had the best IVF effect on the AG129 mice. The average fertilization rate was (43. 35 ± 1. 76)%, and the embryo status was good. All recipient mice became pregnant and gave birth successfully after the embryos were transferred. Conclusions A GSH concentration of 1 mmol / L significantly improves the IVF efficiency of AG129 mice. This provides a potential solution for the biological purification and rapid propagation of mice with IVF difficulties.