One of the major roles of Sertoli cells is to establish the blood-testis (Sertoli cell) barrier (BTB), which is permanently assembled and disassembled to accommodate the translocation of leptotene spermatocytes from the basal into the adluminal compartment of the seminiferous epithelium and to guarantee completion of meiosis and spermiogenesis. Recently, we have demonstrated spermatogenesis to be arrested before spermatid elongation in Gnpat-null mice with selective deficiency of ether lipids (ELs) whose functions are poorly understood. In this study, we have focused on the spatio-temporal expression of several BTB tight-junctional proteins in the first wave of spermatogenesis to obtain insights into the physiological role of ELs during BTB establishment and dynamics. Our data confirm the transient existence of Russell's intermediate or translocation compartment delineated by two separate claudin-3-positive luminal and basal tight junctions and reveal that EL deficiency blocks BTB remodeling. This block is associated with (1) downregulation and mistargeting of claudin-3 and (2) impaired BTB disassembly resulting in deficient sealing of the intermediate compartment as shown by increased BTB permeability to biotin. These results suggest that ELs are essential for cyclic BTB dynamics ensuring the sluice mechanism for leptotene translocation into the adluminal compartment.