The disruption of the gut microbiota by treatment with an antibiotic cocktail (ABx) can trigger an imbalance in immune homeostasis. However, whether the changes in the intestinal microbiota always correspond to the changes in the physiology and immune homeostasis of the host remains unclear. Here, we analyzed the effects of ABx on immune homeostasis by analyzing the colonic transcriptome with 16S rRNA analysis of the gut microbiota on the 7th and 21st days of continuous treatment with ABx. Our results showed that the composition profile of the gut microbiota was similar after 7 and 21 days of ABx treatment. However, after 21 days of ABx treatment, the intestinal inflammation did not deteriorate further. Instead, the inflammation of the host was relieved, and half of the differentially expressed genes in the colon were restored compared with the 7 days of ABx treatment. Furthermore, the enrichment and network analysis of these restored genes indicated that expression of regenerating islet-derived protein 3 beta (Reg3b) and expression of regenerating islet-derived protein 3 gamma (Reg3g), especially Reg3b, may participate in the regulation of the inflammatory response and affect the changes in host immune homeostasis during continuous ABx treatment. Finally, Spearman's correlation analysis showed that the expression of Reg3b is correlated with the growth of Escherichia-Shigella. Our data demonstrated that even though the disruption of the gut microbiota profile induced by ABx treatment is similar, the host response and immune status will be different at different times. Key Points center dot Host immune status can change in different ABx treatment times. center dot Gut microbiota showed same exhaustion state in different ABx treatment times. center dot Host tried to revert to a certain extent after long-term ABx treatment. center dot Reg3b may affect the changes in host immune homeostasis during continuous ABx treatment. center dot The expression of Reg3b correlated with the growth of Escherichia-Shigella.