Emerging evidence suggests that NLRP3 inflammasome was associated with various kinds of immunological diseases including colitis. However, there are few drugs targeting inflammasomes in the treatment of colitis. Several flavonoids have been found to affect the inflammasome pathway, but the mechanism is still confusing. Here we report that VI-16, a synthetic flavonoid compound, exerts potent anti-inflammatory effects on macrophages in DSS-induced colitis mice, which intervened in the activation of NLRP3 inflammasome without affecting intestinal epithelial cells. However, the protection of VI-16 against DSS-induced colitis was dependent on NLRP3 expression in hematopoietic cells. Furthermore, this inhibitory effect of VI-16 was found to be at least partially achieved by decreasing the mitochondrial ROS generation without affecting autophagy. Further studies confirm that VI-16 inhibits the binding of Txnip to NLRP3 by reducing oxidative stress and ultimately inhibits NLRP3 inflammasome. This demonstrates the ability of VI-16 to inhibit the NLRP3 inflammasome activation and its potential use in the treatment of inflammatory bowel disease.