Background: There is increasing evidence that the intestinal microbiota plays a crucial role in the maturation of the immune system and the prevention of diseases during childhood. Early-life short-course antibiotic use may affect the progression of subsequent disease conditions by changing both host microbiota and immunologic development. Epidemiologic studies provide evidence that early-life antibiotic exposures predispose to inflammatory bowel disease (IBD). Methods: By using a murine model of dextran sodium sulfate (DSS)-induced colitis, we evaluated the effect on disease outcomes of early-life pulsed antibiotic treatment (PAT) using tylosin, a macrolide and amoxicillin, a beta-lactam. We evaluated microbiota effects at the 16S rRNA gene level, and intestinal T cells by flow cytometry. Antibiotic-perturbed or control microbiota were transferred to pups that then were challenged with DSS. Results: A single PAT course early-in-life exacerbated later DSS-induced colitis by both perturbing the microbial community and altering mucosal immune cell composition. By conventionalizing germ-free mice with either antibiotic-perturbed or control microbiota obtained 40 days after the challenge ended, we showed the transferrable and direct effect of the still-perturbed microbiota on colitis severity in the DSS model. Conclusions: The findings in this experimental model provide evidence that early-life microbiota perturbation may increase risk of colitis later in life.