Updated project metadata. Acephalic spermatozoa syndrome is severe teratozoospermia, displaying the separation of the sperm head and tail. In 2015, we first reported that genetic ablation of SPATA6 results in the separation of sperm head and tail. Here, we focus on a co-chaperone protein BAG5 that interacts with SPATA6 and expresses in steps 9-16 spermatids. The deficiency of BAG5 in male mice causes the abnormal assembly of the head-tail coupling apparatus (HTCA), leading to acephalic spermatozoa and male infertility. In vivo and in vitro experiments demonstrated that HTCA formation-related proteins (SPATA6), cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1 and DNAL1) were misfolded after loss of BAG5. Further, BAG5 interacts with HSPA8 to regulate its affinity with SPATA6, myosin proteins and dynein proteins. Therefore, we speculate that BAG5 regulates the assembly of the head-tail coupling apparatus (HTCA) by activating the protein-folding function of HSPA8.