Escherichia coli (E. coli) is a common pathogen in veterinary clinical infections, typically not causing disease. However, under certain conditions, it can cause severe diarrhea, septicemia, and respiratory infections in domestic animals. Here, we report a large-scale mortality event in young donkeys, caused by E. coli infection in a farm with a herd of 340 donkeys, of which over 100 were under 4 months old. The characteristics of the E. coli strain were determined through clinical observation, complete blood count, blood biochemical analysis, H&E staining, histopathological examination, 16S rRNA analysis, and antibiotic sensitivity testing. A mice experiment using the isolated bacteria was also conducted, resulting in the onset of clinical and pathological symptoms similar to those observed in the donkeys. Molecular Evolutionary Genetic Analysis (MEGA) showed that the isolated bacteria, named CEG-GZL20, shared 99.98% sequence identity with E. coli. CEG-GZL20 exhibited high antibiotic resistance and only showed sensitivity to two antibiotics, Tetracycline and Gentamicin. As E. coli is a key component of the gut microbiota, responsible for digestion, nutrient absorption, and immune health, diseases and mortality caused by E. coli have significant implications for animal husbandry and human health.