Inflammatory responses elicited by LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is induced by a wide variety of stress signals including infectious agents and cellular disorders. E. coli O157:H7 causes serious gastrointestinal diseases that results in severe inflammation and oxidative stress, causing host cell damage. In this study, we found that E. coli O157:H7 infection induced NLRP3 assembly, caspase-1 activation and interleukin (IL)-1β and IL-18 release in Caco-2 cells. Infection also resulted in mitochondrial dysfunction with disrupted mitochondrial potential and mitochondrial complex-I activity, as well as the cytosolic release of cytochrome c and altered mitochondrial respiratory chain. The damage of mitochondria led to increased production of reactive oxygen species (ROS) and cytosolic release of mitochondrial DNA. Moreover, ROS was required for E. coli O157:H7 induced NLRP3 assembly as inhibiting mitochondrial ROS release by ROS scavengers Mito-TEMPO and N-acetylcysteine abrogated NLRP3 inflammasome activation in Caco-2 cells in response to E. coli O157:H7. Quercetin, one of the most important flavonoids in plant origin foods, had a protective role in inhibiting NLRP3 activation upon E. coli O157:H7 infection by protecting mitochondrial integrity and inhibiting mitochondrial ROS release. In addition, E. coli O157:H7 infection inhibited the host autophagy while quercetin treatment augmented autophagy activation, which further blocked ROS generation and IL-1β and IL-18 release. In summary, E. coli O157:H7 infection induced mitochondrial ROS release and NLRP3 assembly in host cells, while quercetin exerted a preventive role in host cells upon E. coli O157:H7 infection partially due to prevention of ROS production and activation of autophagy.