3Sertoli cells are crucial for spermatogenesis in the seminiferous epithelium because 2 4 their actin cytoskeleton supports vesicle transport, cell junction, protein anchoring and 2 5 spermiation. Here, we show that junction-mediating and regulatory protein (JMY), an 2 6 actin regulating protein, also affects endocytic vesicle trafficking and Sertoli cell 2 7 junction remodeling since disruption of these functions induced male subfertility in 2 8Sertoli cell-specific Jmy knockout mice. Specifically, these mice have: a) impaired 2 9 BTB integrity and spermatid adhesion in the seminiferous tubules; b) high incidence 3 0 of sperm structural deformity; c) reduced sperm count and poor sperm motility. 3 1 Moreover, the cytoskeletal integrity in Sertoli cell-specific Jmy knockout mice was 3 2 compromised along with endocytic vesicular trafficking. These effects impaired 3 3 junctional protein recycling and reduced Sertoli cell junctions. In addition, JMY 3 4 interaction with α -actinin1 and Sorbs2 was related to JMY activity and in turn actin 3 5 cytoskeletal organization. In summary, JMY affects control of spermatogenesis 3 6 through regulating actin filament organization and endocytic vesicle trafficking in 3 7 Sertoli cells. 3 8 3 9