Many studies have shown that antibiotic therapy can attenuate colitis in IL10-deficient (IL10-/-) mice. However, these results have indicated that antibiotics were more successful in preventing, rather than treating established colitis. Those antibiotic treatments attempted to only partially alter the intestinal microbiota and to not eliminate it completely. Therefore, we treated IL10-/- mice with the multiantibiotic regimen that was used to develop a pseudo-germ-free mouse model to determine whether multi-antibiotics attenuated or exacerbated colitis. We evaluated the colitis in IL10-/- mice receiving the antibiotic treatment versus those receiving the water control; furthermore, we investigated the gut microbiota, the intestinal immune cell proportions and the cytokine secretion. Surprisingly, the IL10-/- mice receiving the antibiotic treatment had more severe intestinal colitis and a swollen cecum than those receiving the water control. Moreover, the abundance of microbiota and content of short-chain fatty acids (SCFAs) in the colon were dramatically decreased. Additionally, the proportions of Treg cells and Th1 cells in the colons of IL10-/- mice were also decreased. The mechanism may be that the decrease in the microbiota leads to a decrease in the proportions of Treg cells and SCFAs, which are necessary to maintain intestinal homeostasis. All changes lead to further exacerbated colitis in IL10-/- mice with antibiotic treatment.