In mammalian testes, the blood–testis barrier (BTB), created by specialized junctions between Sertoli cells near the basement membrane of the seminiferous epithelium, provides an indispensable immune-privileged microenvironment for spermatid development. However, the BTB must experience restructuring during the epithelial cycle to facilitate the transit of preleptotene spermatocytes upon the testosterone-induced new TJ fibrils forming behind these cells, which is intimately related to the extensive dynamics of junction protein complexes between Sertoli cells. As key regulators of protein traffic, Rab GTPases participate in delivery of proteins between distinct cellular sites and cross talk with proteins that constitute tight junction and adherens junction. Using primarily cultured Sertoli cells in vitro with an established tight junction permeability barrier that mimics the BTB in vivo, RAB13 was shown to decrease during the testosterone-induced TJ integrity enhancement, accompanied with an increment in protein kinase A (PKA) activity. Furthermore, knockdown of Rab13 was found to resemble the effect of testosterone on Sertoli cell TJ permeability by reinforcing filamentous actin and occludin distribution at the cell–cell interface and promoting the direct interaction between ZO-1 and occludin. Interestingly, the effects of testosterone and Rab13 knockdown on Sertoli cell epithelium were revealed to be antagonized by PKA activity inhibition. In summary, RAB13 serves as a regulatory component in the assembly and restructuring of the TJ fibrils between adjacent Sertoli cells.