Antibiotics (AB), and more particularly penicillins, are the most frequently dispensed drugs in pediatric patients (1) and there is currently increasing concern that AB exposure early in life may have long-term consequences for health. Epidemiological studies have revealed that early life AB exposure increases the risk of diseases that may persist into adulthood such as allergies, inflammatory bowel diseases and obesity (2). Experimental models have shown that the critical period for antibiotic-driven shift in gut microbiota to alter immune and metabolic responses occurs between birth and weaning (3,4). The effects of AB on brain and behavior have been demonstrated in previous studies (5-7) by using high doses of cocktails of mainly broad-spectrum antibiotics administered to adolescent or adult rodents. In this study, we have investigated the long-term effects on gut microbiota, brain and behavior of a clinically relevant dose of oral penicillin given early in life, to both male and female Balb/c mice. Pregnant dams received penicillin V in drinking water 1 week before delivery and until weaning. Penicillin is absorbed by the gastro-intestinal tract, crosses the placenta and is found in the breast milk. The pups therefore received penicillin in utero and during the first 3 weeks of life while nursing. At weaning, pups were separated from their mothers and received regular drinking water. At 6-weeks old, the offspring (n = 72) were subjected to a battery of behavioral tests and gut (ileum, colon) and brain (hippocampus, frontal cortex) tissues were collected after the last test and processed for qPCR and western-blot analysis. Feces were collected at 3-weeks old and 6-weeks old. We found that early life AB exposure had lasting effects on gut microbiota composition, modified the tight junctions of the blood-brain barrier, induced inflammation in the frontal cortex and was associated with changes in brain neurochemistry (Crhr2, Bdnf, Avpr1b). Also, AB-treated mice exhibited decreased anxiety-like behavior, reduced social behavior and preference for social novelty as well as an unexpected aggressive behavior. Supplementation with Lactobacillus rhamnosus JB-1TM during AB treatment restored certain biological and behavioral parameters. This study revealed that clinical dose penicillin given early in life to mice had long-term effects on behavior, brain inflammation and gut microbiota, and may raise concerns about the long-term behavioral consequences of AB therapy.