Escherichia coli (E. coli) O₁-induced diarrhea is associated with intestinal microbial imbalance, however, the results of using oral antibiotics still remain poor. To overcome such problem, our study investigates the role of metabolites from stable flies (MSF) in the occurrence of diarrhea. The amino acid composition and molecular weight analysis of MSF by RP-HPLC and GPC, respectively. Besides the normal control group, SPF mice in other group were inoculated with E. coli O1 received treatment as follows over a period of 7 days saline solution (E. coli control), ciprofloxacin (0.13 g/kg; positive control) and MSF (2, 4 and 8 mg/kg) dosage. Throughout the experiment, defecation and body weights were examined and recorded. On the eighth day, after administering anesthesia, blood, tissue of small intestine samples were obtained for immunological and anti-oxidant. Small intestinal tissues and cecum contents samples were used for histopathological and 16S rDNA sequencing analysis. Our showed that MSF was rich in isoleucine, and its molecular weight less than 400 Da is 60.03%. MSF (4 and 8 mg/kg) and ciprofloxacin, significantly decreased IL-6, IL-8 and TNF-α levels, whereas, increased IL-2, IL-4, IL-10, INF-γ, IgA and IgG levels in mice having diarrhea. These treatments also reversed intestinal flora imbalance as indicated by the increased in Firmicutes-to-Bacteroidetes ratio and Clostridium levels (P < 0.05) and improved 5-HT, CAT and SOD levels. MSF favored diarrhea management as compared to ciprofloxacin, suggesting that MSF can be used in the management of E. coli O₁-induced diarrhea, in normal gut microbiota and normal intestinal antioxidant function.