The H9N2 subtype of avian influenza virus (AIV) is common in poultry production. It causes mild clinical signs but rarely leads to poultry mortalities. However, higher mortality can occur in chickens with co-infections, especially avian pathogenic Escherichia coli (APEC), which results in huge economic losses for the poultry industry. Unfortunately, the mechanism of co-infection remains unknown. Our previous studies screened several proteins associated with bacterial adhesion, including transforming growth factor beta-1 (TGF-β1), integrins, cortactin, E-cadherin, vinculin, and fibromodulin. Herein, we investigated the contribution of TGF-β1 to APEC adhesion after H9N2 infection. We first infected H9N2 and APEC in chicken, chicken embryo and DF-1 cells, and demonstrated that H9N2 infection promotes APEC adhesion to hosts in vitro and in vivo by plate count method. Through real-time fluorescence quantification and enzyme-linked immunosorbent assay, it was demonstrated that H9N2 infection not only increases TGF-β1 expression but also its activity in a time-dependent manner. Then, through exogenous addition of TGF-β1 and overexpression, we further demonstrated that TGF-β1 can increase the adhesion of endothelial cells to DF-1 cells. Furthermore, the capacity of APEC adhesion to DF-1 cells was significantly decreased either by adding a TGF-β1 receptor inhibitor or using small interfering RNAs to interfere with the expression of TGF-β1. To sum up, H9N2 infection can promote the upregulation of TGF-β1 and then increase the adhesion ability of APEC. Targeting TGF-β1 and its associated pathway will provide valuable insights into the clinical treatment of E. coli secondary infection induced by H9N2 infection.