Mycotoxin zearalenone (ZEN) is a cereal contaminant produced by various species of Fusarium fungi. When interacting with estrogen receptors, ZEN leads to animal fertility disturbances and other reproductive pathologies. Few data are available on the effects of perinatal exposure to ZEN, particularly in the blood-testis barrier. The aim of this study was to assess the impact of ZEN in adult rats exposed neonatally. We focused on the expression and cellular localization of major ABC transporters expressed in adult rat testis, comparing ZEN effects with those of Estradiol Benzoate (EB) neonatal exposure. Dose-dependent and long term modulations of mRNA and protein levels of Abcb1, Abcc1, Abcg2, Abcc4 and Abcc5 were observed, along with Abcc4 protein cellular delocalization. ZEN exposure of SerW3 Sertoli cells showed modulation of Abcb1, Abcc4 and Abcc5. Comparison with EB exposure showed similar modulation profiles for Abcg2 but differential modulations for Abcb1, Abcc1, Abcc4 and Abcc5 in vivo, and a similar profile for Abcb1 modulation by ZEN and EB, but differential modulation for Abcc4 and Abcc5 in vitro. ZEN and EB effects were inhibited by in vitro addition of the pure anti-estrogen ICI 182.780, suggesting the at least partial implication of ZEN estrogenic activity in these modulations. These results suggested that ZEN neonatal exposure could affect the exposure of testis to ABC transporter substrates, and negatively influence spermatogenesis and male fertility.