Genistein (GEN) is a natural polyphenolic compound widely present in leguminous plants, which has many biological functions such as anti-inflammatory and antioxidant activities, and has attracted attention in the treatment of inflammatory bowel disease (IBD). However, the molecular mechanism underlying the beneficial effects of GEN in IBD remains unclear. Here, we demonstrated that GEN enhanced the relative abundance of beneficial bacteria (e.g., Akkermansia muciniphila) and increased microbiota-derived short-chain fatty acids (SCFAs) levels in colitis mice. Further, the antibiotic cocktails (ABX) and fecal microbiota transplantation (FMT) treatments confirmed that gut microbiota at least partially mediated the anti-colitis effect of GEN. Interestingly, we found that GEN could also activate G protein-coupled receptor 30 (GPR30) and its downstream transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in intestinal epithelial cells (IECs). The activation of the GPR30-Nrf2 signaling led to reduced reactive oxygen species (ROS) production, which consequently inhibited NLRP3 inflammasome activation and improved intestinal epithelial barrier dysfunction. In addition, studies using GPR30 knockout mice confirmed that GPR30 is crucial for inhibiting NLRP3 inflammasome activation and alleviating colitis. Collectively, our study unveils that GEN is an effective anti-inflammatory nutrient and suggests that both the gut microbiota and the GPR30-Nrf2 signaling pathway represent potential therapeutic targets for treating IBD.