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Escherichia coli (E. coli) infection is very common among young growing animals, and zinc supplementation is often used to alleviate inflammation induced by this disease. Therefore, the objective of this study was to evaluate whether chitosan- chelated zinc (CS-Zn) supplementation could attenuate gut injury induced by E. coli challenge and to explore how CSZn modulates cecal microbiota and alleviates intestinal inflammation in weaned rats challenged with E. coli. 36 weaned rats (55.65 ± 2.18 g of BW, n = 12) were divided into three treatment groups consisting of unchallenged rats fed a basal diet (Control) and two groups of rats challenged with E. coli and fed a basal diet or a diet containing 640 mg/kg CS-Zn (E. coli + CS-Zn, containing 50 mg/kg Zn) for a 14-day experiment. On days 10 to 12, each rat was given 4 ml of E. coli solution with a total bacteria count of 1010 CFU by oral gavage daily or normal saline of equal dosage. CS-Zn supplementation mitigated intestinal morphology impairment (e.g. higher crypt depth and lower macroscopic damage index) induced by E. coli challenge (P < 0.05), and alleviated the increase of Myeloperoxidase (MPO) activity after E. coli challenge (P < 0.05). 16S rRNA sequencing analyses revealed that E. coli challenge significantly increased the abundance of Verrucomicrobia and E. coli (P < 0.05). However, CS-Zn supplementation increased the abundance of Lactobacillus and decreased the relative abundance of Proteobacteria, Desulfovibrio and E. coli (P < 0.05). The concentrations of butyrate in the cecal digesta, which decreased due to the challenge, were higher in the E. coli + CS-Zn group (P < 0.05). In addition, CS-Zn supplementation significantly prevented the elevation of pro-inflammatory cytokines IL-6 concentration and upregulated the level of anti-inflammatory cytokines IL-10 in cecal mucosa induced by E. coli infection (P < 0.05). In conclusion, these results indicate that CS-Zn produces beneficial effects in alleviating gut mucosal injury of E. coli challenged rats by enhancing the intestinal morphology and modulating cecal bacterial composition, as well as attenuating inflammatory response.