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Colibacillosis, a bacterial disease caused by avian pathogenic E. coli(APEC), is a prevalent condition in the poultry industry, resulting in substantial economic losses annually. Previously, we identified PTENas a crucial candidate gene that may play a significant role in chicken's immune response to APEC infection. Bioinformatics analysis indicated that the PTENprotein was unstable, hydrophilic and nuclear localization, with multiple putative phosphorylation sites and a high degree of similarity to duck and goose PTEN. Moreover, PTENexhibited high expression levels in various tissues such as the stomach, cecum, small intestine, spleen, thymus, harderian gland, muscle, cerebrum, cerebellum, lung, and liver in comparison to heart tissue. Overexpression of PTENresulted in a significant promotion of the expression level of pro-apoptosis genes and inflammatory mediators, as well as the production of NO, with or without APEC infection, which led to cellular injury. Furthermore, overexpression of PTENwas found to regulate the expression levels of autophagy related genes, regardless of APEC infection. Additionally, PTENwas a target gene of gga-miR-20a-5p and regulated by gga-miR-20a-5p upon APEC infection. Taken together, these findings establish a foundation for investigating the biological function of chicken PTEN, providing a potential target for future treatments against APEC infection as well as the breeding of genetically resistant poultry.