Avian pathogenic Escherichia coli (APEC), collectively known as causative agent of extraintestinal infections, is an important cause of morbidity and mortality in poultry. Currently, quorum sensing (QS), biofilm formation and virulence factors are considered as novel prospective targets for antimicrobial therapy to control APEC invasion. In addition, inflammatory responses are also served as the major pathological features of APEC invasion. This study was aimed to explore the effect of baicalin on APEC and APEC-induced inflammatory responses. After treatment with baicalin, we mainly examined the AI-2 secretion, biofilm formation, expression of virulence genes of APEC, and the levels of inflammatory cytokines, as well as the expression of NF-κB pathway. Our results showed that baicalin significantly inhibited the QS via decreasing the AI-2 secretion, biofilm formation, and the expression of virulence genes of APEC such as LsrB, LsrK, LuxS, pfs, H-NS, fimA, fimB, fyuA, csgA, csgB, and rpoS. Moreover, baicalin significantly attenuated the release of lactate dehydrogenase (LDH), and the adhesion of APEC to chicken type II pneumocytes to reduce cell damage. Furthermore, baicalin also inhibited the expression of pro-inflammatory cytokines and NF-κB activation. Thus, our data revealed that baicalin could interfere with the quorum sensing, biofilm formation and virulence genes expression to relieve the APEC pathogenicity. Additionally, baicalin decreased the inflammatory responses of chicken type II pneumocytes induced by APEC. Taken together, these data provide a novel potential pharmaco-therapeutic approach to chicken colibacillosis.