Dioxins, mostly through activation of aryl hydrocarbon receptor (AhR), are potent toxic substances widely distributed in the environment, while moderated suppression of AhR also exhibits anti-tumor effects. Therefore, the proper modulation of AhR activity may counteract AhR-mediated toxicities and certain diseases. In this investigation, we identified several novel AhR moderate agonists and antagonists using chemical biology approaches. The mechanisms and mode of interactions with AhR by these hits were also revealed using both experimental and computational studies. The newly identified AhR moderate agonists and antagonists were predicted to bind to AhR and modulate AhR signaling. The structure-activity relationships of moderate agonists and antagonists and their unique binding features with AhR have created a solid framework for further optimization of the next generation of AhR modulators.