The estrogenic chemical nonylphenol (NP) and the antiandrogenic agent di-n-butyl phthalate (DBP) are regarded as widespread environmental endocrine disruptors (EEDs) which at high doses in some species of laboratory animals have adverse effects on male reproduction and development. Given the ubiquitous coexistence of various classes of EEDs in the environment, their combined effects warrant investigation. In this study, we attempted to clarify the interactions of NP and DBP on tight junctions (TJs) between rat Sertoli cells. In the in vitro experiment, monobutyl phthalate (MBP), the active metabolite of DBP, was used instead of DBP. Sertoli cells were isolated from Sprague-Dawley rats, and treated with NP and MBP, singly or combined. The morphology of Sertoli cells, and structure and functionality of TJs were measured. In the in vivo experiment, rats were gavaged on postnatal day 23–35 with a single or combined NP and DBP treatment. Testicular weight and morphology of TJs were recorded. These data indicated that NP and DBP/MBP, either in single or in combination, induced the structural and function changes of Sertoli cell tight junctions, both in vivo and in vitro. The combined effect on the regulation of TJ proteins at both the protein and gene levels was correlated to the effect exerted by NP, suggesting that the structure and function of Sertoli cells were more sensitive to exposure to NP than MBP.