Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell-cell and Sertoli cell-spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell-cell interface, and the apical ES at the Sertoli-spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.