During spermatogenesis, the molecular mechanism that confers spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES), a testis-specific F-actin-rich adherens junction, in the rat testis remains elusive. Herein, the activated form of focal adhesion kinase (FAK), p-FAK-Tyr397, a component of the apical ES that expressed stage-specifically in stage VII-early stage VIII tubules, was found to be a crucial apical ES regulator. Using an FAK-Y397E phosphomimetic mutant cloned in a mammalian expression vector for its transfection versus FAK in adult rat testes in vivo, its overexpression was found to cause defects in spermiation. These defects in spermiation was manifested by entrapment of spermatids in the seminiferous epithelium in late stage VIII-X tubules, and were mediated by a disruption on the spatiotemporal expression and/or mis-localization of actin regulatory proteins, Arp3 (actin-related protein 3 that induces branched actin polymerization), Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and palladin (an actin cross-linking and bundling protein); as well as adhesion proteins nectin-2 and nectin-3. This thus perturbed changes of F-actin organization at the apical ES to facilitate spermiation, which also led to a concomitant alteration in the distribution and up-regulation of nectin-2 and nectin-3 at the apical ES. As such, nectin-2 and -3 remained at the apical ES to anchor step 19 spermatids to the epithelium via F-actin to confer adhesion, delaying spermiation. These findings illustrate a mechanistic pathway mediated by p-FAK-Tyr397 that regulates spermatid adhesion at the apical ES in vivo.