During spermatogenesis, leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) to gain entry into the adluminal compartment for further development. At the same time, these as well as other germ cell types in the epithelium must retain their close association with Sertoli cells via specialized cell junctions. In this study, we demonstrate that RAB13—a guanosine triphosphatase (GTPase) known to participate in tight junction function in other epithelia—also participates in the dynamics of the ectoplasmic specialization, a testis-specific type of anchoring junction. By immunohistochemistry microscopy, RAB13 localized to the ectoplasmic specialization. Moreover, RAB13 was found to associate with vinculin (VCL) and espin (ESPN), two putative ectoplasmic specialization actin (ACT)-binding proteins, by coimmunoprecipitation and immunofluorescence microscopy experiments. To address the role of RAB13 in ectoplasmic specialization dynamics, an in vivo model was used in which administration of Adjudin induced the disassembly of Sertoli-germ cell anchoring junctions. Following administration of this drug, the RAB13 level decreased steadily when the loss in testicular weight was taken into account. Similarly, the association of RAB13 with VCL decreased but was not completely lost during Adjudin-mediated ectoplasmic specialization restructuring. Taken collectively, these results suggest that RAB13 functions in ectoplasmic specialization dynamics in the testis.