Avian influenza A (H9N2) avian influenza is a low-pathogenic avian influenza circulating in poultry and wild birds worldwide and frequently contributes to chicken salpingitis that is caused by avian pathogenic Escherichia coli (APEC), leading to huge economic losses and risks for food safety. Currently, how the H9N2 virus contributes to APEC infection and facilitates salpingitis remains elusive. In this study, in vitro chicken oviduct epithelial cell (COEC) model and in vivo studies were performed to investigate the role of H9N2 viruses on secondary APEC infection, and we identified that H9N2 virus enhances APEC infection both in vitro and in vivo. To understand the mechanisms behind this phenomenon, adhesive molecules on the cell surface facilitating APEC adhesion were checked, and we found that H9N2 virus could upregulate the expression of fibronectin, which promotes APEC adhesion onto COECs. We further investigated how fibronectin expression is regulated by H9N2 virus infection and revealed that transforming growth factor beta (TGF-β) signaling pathway is activated by the NS1 protein of the virus, thus regulating the expression of adhesive molecules. These new findings revealed the role of H9N2 virus in salpingitis co-infected with APEC and discovered the molecular mechanisms by which the H9N2 virus facilitates APEC infection, offering new insights to the etiology of salpingitis with viral-bacterial co-infections.IMPORTANCEH9N2 avian influenza virus (AIV) widely infects poultry and is sporadically reported in human infections. The infection in birds frequently causes secondary bacterial infections, resulting in severe symptoms like pneumonia and salpingitis. Currently, the mechanism that influenza A virus contributes to secondary bacterial infection remains elusive. Here we discovered that H9N2 virus infection promotes APEC infection and further explored the underlying molecular mechanisms. We found that fibronectin protein on the cell surface is vital for APEC adhesion and also showed that H9N2 viral protein NS1 increased the expression of fibronectin by activating the TGF-β signaling pathway. Our findings offer new information on how AIV infection promotes APEC secondary infection, providing potential targets for mitigating severe APEC infections induced by H9N2 avian influenza, and also give new insights on the mechanisms on how viruses promote secondary bacterial infections in animal and human diseases.