Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality in humans. However, the underlying mechanism(s) remains unknown. Herein, PFOS at 10–20 µM (~5–10 µg/ml) was found to be more potent than BPA (100 µM) in perturbing blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction (TJ)-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin regulatory and adhesion proteins at the cell–cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43 (Cx43), a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr^407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific miRNA, miR-135b, to knockdown the expression of phosphorylated focal adhesion kinase (FAK)-Tyr^407 was found to worsen PFOS-mediated Sertoli cell TJ disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated focal adhesion kinase (FAK)-Tyr^407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mis-localization of actin-regulatory and adhesion proteins at the BTB.