Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract, of which the etiology involves environmental, genetic and microbial factors. Our group and others have shown a high prevalence of the invasive E. coli strains, designated adherent-invasive E. coli (AIEC), in the intestinal mucosa of CD patients. Upon AIEC infection, autophagy is induced in host cells to restrain the replication of the bacteria. The signalling pathway inducing autophagy response to AIEC infection, however, remains unknown. Here, we investigated the role of the GCN2/eIF2a/ATF4 pathway in such mechanism. We found that infection of intestinal epithelial cells (IECs) with an AIEC reference strain LF82 increased the levels of phospho-GCN2, phospho-eIF2a and enhanced ATF4 protein expression. The later consequently led to upregulated mRNA expression levels of target genes of ATF4, such as ASNS, TRB3, GLYT, CHOP, ATF3, and autophagy genes (MAP1LC3B, p62). Intracellular multiplication of AIEC was increased in GCN2-/- and ATF4-/- mouse embryonic fibroblasts (MEFs) compared with that in wild type MEFs as determined by bacterial invasion assay and confocal microscopy using a LF82-GFP strain. These results were validated in vivo using GCN2 knockout mice and a model of AIEC infection that we previously established. AIEC infection induced a stronger pro-inflammatory response in GCN2-/- and ATF4-/- MEFs than in wild type MEFs, and in GCN2 knockout mice versus wild type mice. Autophagy induction in response to infection, assessed by Western blot and immunofluorescent analyses of LC3 levels, was decreased in GCN2-/- and ATF4-/- MEFs compared with wild type MEFs. In conclusion, our study shows that upon AIEC infection, the CN2/eIF2a/ATF4 signaling pathway is activated in host cells, which is served as a host defense mechanism to induce a functional autophagy-mediated control of AIEC intracellular replication.