We previously established a spontaneously occurring monoclonal antibody, namely Ts3, that was reactive to sperm from an aged male mouse. The present study investigated the characteristic properties and reproductive functions of Ts3. Immunofluorescent staining revealed that Ts3 reacted to epididymal sperm, and the corresponding antigen was located in the midpiece and principal piece. Immunohistochemistry revealed positive reactions in the germ cells and Sertoli cells in the testis, the epithelial cells in the epididymis and vas deferens. Through western blotting with two-dimensional electrophoresis, we demonstrated that Ts3 reacted with four spots, which were around Mr ∼25,000-60,000 and pI 5-6. MALDI-TOF/TOF mass spectrometry identified outer dense fiber 2 (ODF2) as a candidate for Ts3. ODF2 is a cytoskeletal structural component located in the midpiece and principal piece of the flagella of mammalian sperm. This was validated with the result of immunofluorescent staining, suggesting that ODF2 was the main target antigen for Ts3. Sperm immobilization test showed that Ts3 possessed sperm immobilizing activity. Furthermore, Ts3 impaired early embryo development but not in vitro fertilization. These results suggest that ODF2 plays an important role in both sperm function and early embryonic development.