Sperm express the Toll-like receptor (TLR) family and have an innate immune function defending against bacterial infection in semen. It has been known that virus infection is also observed in semen with low motility of sperm, suggesting that sperm are also able to respond to virus infection. However, there is little information about the expression and roles of TLRs that recognize virus-released pathogens in semen. The present study clearly showed that sperm express functional TLR9 that recognizes the unmethylated CpG DNA sequence. Using a specific antibody, we detected the expression of TLR9 in sperm. However, the positive band was disappeared by progesterone treatment that induced the acrosome reaction. When sperm were cultured with the TLR9 ligand, sperm motility significantly decreased in a time-dependent manner. The treatment of sperm with the TLR9 ligand affected the acrosomal status and suppressed the BSA-induced capacitation of sperm. Additionally, sperm were used for in vitro fertilization and injection into the uterus of super-ovulated female mice following pre-culture with the TLR9 ligand. The fertilization rates were significantly suppressed both in vitro and in vivo. From these results, we conclude that sperm express and have functional TLR9 that potentially recognizes virus infection in semen.