Abstract: Objective To explore the function of seminal vesicle gland in the process of fertilization. Methods C57BL/6 male mice (8 weeks) were taken to establish seminal vesicle gland exposure mice (a control group) and seminal vesicle gland resection mice (an experiment group). Then, the testes were weighted and observed, and the morphological structure of the testicular lumen were evaluated by hematoxylin-eosin staining. The number and motility of sperms were detected by computer-assisted semen analysis (CASA). Mice in both groups were mated with C57BL/6 female mice at a ratio of 1∶1 and maintained for six months to observe the number of litters. The female mice were mated with mice in both groups at a ratio of 2∶1 and the resultant embryos were taken six days later for photography. Furthermore, female mice were mated with mice in both groups at a ratio of 1∶1 for in vivo fertilization and the resultant pronuclei were obtained by surgery on the next day to observe the development of the embryos in vitro. Results Compared with the control group, mice in the experiment group showed no significant differences in morphology, weight, and testicular lumen structure (P>0.05). There were also no statistical difference in sperm motility and counts between the two groups (P>0.05). According to the fertility test, mice in the experiment group were sterile, while those in the control group had about 7 litters on average. Furthermore, embryos were not found in female mice mated with mice in the experiment group.In vivo fertilization experiments showed that part of sperms from mice in the experiment group completed fertilization to form pronuclei after seminal vesicle gland removal, which however were unable to develop into normal 4 cells. Conclusions Part of the sperms from vesicle gland-removed mice can complete fertilization and develop into 2 cells, but are unable to form the normal 4 cells, and eventually leads to infertility.