The redox state of thiol groups derived from cysteine residues in proteins regulates cellular functions. Changes in the redox state of thiol groups in the epididymis are involved in sperm maturation. Furthermore, the redox state of thiol groups in proteins changes during the process of sperm capacitation. However, the effect of the redox state of thiol groups in sperm membrane proteins on the fertilization ability of sperm has not been studied. Therefore, in this study, we oxidized thiol groups in sperm membrane proteins using 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), which is a thiol-selective oxidizing agent, and examined the effect of oxidation of these thiol groups on the fertilization ability of sperm. Oocytes and sperm were obtained from C57BL/6 J mice, and Jcl:ICR mice were used as recipients for embryo transfer. Oxidation of the thiol groups by DTNB decreased the in vitro fertilization rate, and removal of the zona pellucida recovered the fertilization rate. DTNB treatment decreased the amplitude of the lateral head, which is an indicator of hyperactivation, and suppressed an increase in the intracellular calcium ion concentration, which is essential for hyperactivation. These findings suggest that oxidation of thiol groups in sperm membrane proteins can decrease the fertility of sperm by suppressing calcium ion influx and hyperactivation.