During spermatogenesis, immature spermatocytes traverse the blood–testis barrier (BTB) and enter the apical apartment of seminiferous epithelium for further development. This course involves extensive junction disassembly and reassembly at the BTB. P-glycoprotein is known to be coded by two genes in rodents, namely Abcb1a and Abcb1b. Our previous studies showed that simultaneously silencing Abcb1a and Abcb1b genes in Sertoli cells impeded BTB integrity. However, the individual role of Abcb1a and Abcb1b in regulating BTB dynamics remains uninvestigated. Here, single knockdown of Abcb1a by RNAi impeded the in vitro Sertoli cell permeability barrier via redistributing TJ proteins, accelerating endocytosis, and affecting endocytic vesicle-mediated protein transportation that undermined Sertoli cell barrier. F5-peptide model was used to induce cell junction disruption and subsequent restructuring in primary Sertoli cells. F5-peptide perturbed this barrier, but its removal allowed barrier ‘resealing’. Abcb1b knockdown was found to inhibit barrier resealing following F5-peptide removal by suppressing the restore of the expression and distribution of junction proteins at BTB, and reducing the migration of internalized junction proteins back to Sertoli cell interface. In summary, Abcb1a is critical in maintaining BTB integrity, while Abcb1b is crucial for junction reassembly at the BTB.