A number of sperm proteins are involved in the processes from gamete adhesion to fusion, but the underlying mechanism is still unclear. Here, we established a mouse mutant, the EQUATORIN-knockout (EQTN-KO, Eqtn-/-) mouse model and found that the EQTN-KO males have reduced fertility and sperm-egg adhesion, while the EQTN-KO females are fertile. Eqtn-/- sperm were normal in morphology and motility. Eqtn-/--Tg (Acr-Egfp) sperm, which were produced as the acrosome reporter by crossing Eqtn-/- with Eqtn+/+-Tg(Acr-Egfp) mice, traveled to the oviduct ampulla and penetrated the egg zona pellucida of wild-type females. However, Eqtn-/- males mated with wild-type females showed significant reduction in both fertility and the number of sperm attached to the zona-free oocyte. Sperm IZUMO1 and egg CD9 behaved normally in Eqtn-/- sperm when they were fertilized with wild-type egg. Another acrosomal protein, SPESP1, behaved aberrantly in Eqtn-/- sperm during the acrosome reaction. The fertility impairment of EQTN/SPESP1-double KO males lacking Eqtn and Spesp1 (Eqtn/Spesp1-/-) was more severe compared with that of Eqtn-/- males. Eqtn-/--Tg (Eqtn) males, which were generated to rescue Eqtn-/-males, restored the reduced fertility.