Chlorostyrenes (CSs) are primarily derived from industrial by-products and are persistent and accumulative in the environment. In this study, the estrogenic and antiestrogenic activities of CSs (o-CS, m-CS, p-CS, DiCS, octa-CS) were evaluated using in vitro bioassays. o-CS and octa-CS have both estrogenic and antiestrogenic activity in the E-SCREEN assay and the ERE-reporter gene assay, indicating effects on a classical ER-mediated pathway. m-CS showed estrogenic activity in E-SCREEN but not in ERE-reporter gene assays, indicating that it may work through a non-classical ER-mediated pathway. Finally, DiCS only showed antiestrogenic activity via an ER-independent pathway, which can be induced by depletion of endogenous E(2) level by the inhibition of aromatase activity and the stimulation of E(2) metabolism. Although CSs have structural similarities to dioxins/furans, they did not have AhR agonist effects. This study is the first to show the estrogenic and antiestrogenic activity of several CSs using in vitro bioassay systems, including whether the compounds work via ER-mediated or/and non-ER-mediated pathways.