INTRODUCTION: Antimicrobial peptides (AMPs) are endogenous peptides that have been identified to alleviate intestinal epithelial barrier inflammation and dysfunction caused by enteropathogenic Escherichia coli (EPEC) infection; nonetheless, the upstream molecular mechanism of the production of AMPs is poorly understood. MATERIAL AND METHODS: The binding of signal transducer and activator of transcription (STAT) 1 (STAT1) to mucin 4 (MUC4) was examined by co-immunoprecipitation assay. To detect the influence of STAT1 and MUC4 expression, a C57BL/6 mouse model of EPEC infection in vivo and an EPEC infected intestinal epithelial cell (IEC) in vitro model were established. Expression levels of STAT1, MUC4, phosphorylated (p)-STAT1, proinflammatory cytokines, zonula occludens-1 (ZO-1) and AMP-related genes in mouse ileum and/or IEC were analyzed by immunohistochemical test, immunofluorescence assay, Western blot, and/or qRT-PCR. Meanwhile, IEC viability and apoptosis were measured using CCK-8 assay and flow cytometry. RESULTS: p-STAT1, MUC4, ZO-1 and AMP-related genes were lowly expressed in the ileum of EPEC-infected mice. p-STAT1 and MUC4 bound to each other. The expression levels of STAT1 and MUC4 were decreased in EPEC-infected IEC. STAT1 overexpression counteracted the EPEC-induced reduction of viability, apoptosis promotion, ZO-1 activity inhibition, release of proinflammatory cytokines, and downregulation of MUC4 and AMP-related genes in IEC. MUC4 knockdown partly counteracted the effect of STAT1 overexpression, but did not affect the forced STAT1 overexpression in EPEC-infected IEC. CONCLUSIONS: STAT1/MUC4 pathway activation promotes AMP production to mitigate intestinal epithelium barrier injury caused by EPEC infection.