Phthalate esters have been extensively used as a plasticizer of synthetic polymers. Previous studies have revealed that some phthalate esters including di(n-butyl) phthalate (DBP) induce spermatogenic cell apoptosis, although its mechanism is not yet clear. The present study describes that disruption of Sertoli cell vimentin filaments by DBP administration may relate to spermatogenic cell apoptosis. The present histopathological study revealed that a single oral administration of 500 mg/kg DBP caused progressive detachment and displacement of spermatogenic cells away from the seminiferous epithelium and sloughing of them into the lumen. Degenerative spermatogenic cells characterized by chromatin condensation were frequently observed in DBP-treated rats. Ultrastructurally, the degenerative spermatogenic cells were separated from their neighbours, and a collapse of Sertoli cell vimentin filaments was recognized in DBP-treated rats. Sertoli cell cultures showed the increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, vimentin filaments clearly showed a gradual collapse in DBP-exposed Sertoli cells in vitro. These in vivo and in vitro experiments indicate that DBP-induced collapse of Sertoli cell vimentin filaments may lead to detachment of spermatogenic cells, and then detached cells may undergo apoptosis because of loss of the support and nurture provided by Sertoli cells.