Adjudin is known to specifically affect Sertoli-germ cell adhesion, resulting in germ cell loss from the seminiferous epithelium and transient infertility. The apical ectoplasmic specialization (apical ES) was shown to be the primary target of adjudin since adhesion was unaffected in organs which lack this structure. Herein, we expand previous findings by treating rat pups with adjudin, and we aim to address two questions. First, can adjudin perturb germ cell adhesion in the seminiferous epithelium of testes in which the apical ES is not yet present? Second, can adjudin affect assembly of the blood–testis barrier (BTB) at 15–18 days of age? Interesting changes were noted when aged-matched testes from control and adjudin-treated rats were examined, including a delay in the appearance of developing germ cells, as well as a delay in the formation of the tubule lumen. Immunoblotting using antibodies against BTB-constituent proteins indicated that formation of the BTB was affected in rat pups gavaged with adjudin. These results were corroborated by immunofluorescence microscopy which showed profound changes in the cellular distribution of tight junction (TJ) and basal ES proteins. Moreover, the BTB was shown to be compromised in 30-day old rats when its integrity was assessed by a functional in vivo assay. By 45 days of age, however, the seminiferous epithelium of treated rats was indistinguishable from that of control rats. Collectively, these results demonstrate that adjudin targets the apical ES, as well as the basal ES and TJ, which in turn delays assembly of the BTB.