The association between prolonged antibiotic (AB) use in neonates and increased incidence of later life diseases is not yet fully understood. AB treatment in early life alters intestinal epithelial cell composition, functioning, and maturation, which could be the basis for later life health effects. Here, we investigated whether AB-induced changes in the neonatal gut persisted up to adulthood and whether early life AB had additional long-term consequences for gut functioning. Mice received AB orally from postnatal day 10 to 20. Intestinal morphology, permeability, and gene and protein expression at 8 weeks were analyzed. Our data showed that the majority of the early life AB-induced gut effects did not persist into adulthood, yet early life AB did impact later life gut functioning. Specifically, the proximal small intestine (SI) of adult mice treated with AB in early life was characterized by hyperproliferative crypts, increased number of Paneth cells, and alterations in enteroendocrine cell-specific gene expression profiles. The distal SI of adult mice displayed a reduced expression of antibacterial defense markers. Together, our results suggest that early life AB leads to structural and physiological changes in the adult gut, which may contribute to disease development when homeostatic conditions are under challenge.