Arctigenin was previously proven to inhibit Th17 cell differentiation and thereby attenuate colitis in mice by down-regulating the activation of mechanistic target of rapamycin complex 1 (mTORC1). The present study was performed to address its underlying mechanism in view of estrogen receptor (ER). The specific antagonist PHTPP or siRNA of ER beta largely diminished the inhibitory effect of arctigenin on the mTORC1 activation in T cell lines and primary CD4(+) T cells under Th17-polarization condition, suggesting that arctigenin functioned in an ER beta-dependent manner. Moreover, arctigenin was recognized to be an agonist of ER beta, which could bind to ER beta with a moderate affinity, promote dissociation of ER beta/HSP90 complex and nuclear translocation and phosphorylation of ER beta, and increase the transcription activity. Following activation of ER beta, arctigenin inhibited the activity of mTORC1 by disruption of ER beta-raptor-mTOR complex assembly. Deficiency of ER beta markedly abolished arctigenin-mediated inhibition of Th17 cell differentiation. In colitis mice, the activation of ER beta, inhibition of mTORC1 activation and Th17 response by arctigenin were abolished by PHTPP treatment. In conclusion, ER beta might be the target protein of arctigenin responsible for inhibition of mTORC1 activation and resultant prevention of Th17 cell differentiation and colitis development.