Simple Summary Zearalenone (ZEN) widely contaminates all the feed crops, and ZEN may cause harmful damage to animals and humans. Different animals have different sensitivity to ZEN. Among these animals, chickens show a strong resistance. Intestinal microorganisms are essential in digestion and degradation. Therefore, we hypothesise whether intestinal microorganisms in chickens play an important role in digesting and degrading ZEN. In this study, we found that intestinal microorganisms could degrade ZEN to a certain degree by both vivo and vitro experiments. We concluded that the intestinal microbiota of broilers had metabolic effects on ZEN and alleviated antioxidant and liver damage caused by ZEN to broilers. Moreover, we found some key bacteria that are important in degrading ZEN. Zearalenone (ZEN) is an estrogenic mycotoxin, and chickens are relatively insensitive to it. In this study, the effects of intestinal microorganisms on ZEN metabolism and toxicity mitigation in broilers were studied by two experiments. Firstly, in vitro, ZEN was incubated anaerobically with chyme from each part of the chicken intestine to study its intestinal microbial metabolism. Then, in vivo, we explored the effects of intestinal microbiota on ZEN by inhibiting intestinal microorganisms. Broilers were fed a control diet, 2.5 mg/kg ZEN diet, microbial inhibition diet or 'microbial inhibition +2.5 mg/kg ZEN' diet. In vitro, the results showed that the rates of ZEN degradation by microorganisms in the duodenum, ileum, caecum, and colon were 56%, 12%, 15%, and 17%, respectively, and the microorganisms could convert ZEN into Zearalenol (ZOL). After microbial inhibition in vivo, the content of ZEN and its metabolites in excreta of broilers increased significantly, and antioxidant damage and liver damage were aggravated. 16S rRNA sequencing results showed that antioxidant indices and the content of ZEN and its metabolites in excreta were significantly correlated with the relative abundance of Streptococcus, Lactococcus and Enterococcus, etc. In conclusion, the intestinal microorganisms of broilers play an important role in ZEN metabolism and ZEN-induced antioxidant and liver injury mitigation, among which the key bacteria include Streptococcus, Lactococcus and Enterococcus, etc.