There is increasing awareness of the need to consider potential long-term effects of antibiotics on the health of children. In addition to being associated with immune and metabolic diseases, there is evidence that early-life antibiotic exposure can affect neurodevelopment. Here we investigated the effect of low dose of penicillin V on mice when administered for 1 week immediately prior to weaning. We demonstrated that exposure to the antibiotic during the pre-weaning period led to long-term changes in social behaviour, but not anxiety-like traits, in male mice only. The change in behaviour of males was associated with decreased hippocampal expression of AVPR1A and AVPR1B while expression of both receptors was increased in females. Spleens of male mice also showed an increase in the proportion of activated dendritic cells and a corresponding decrease in regulatory T cells with penicillin exposure. All changes in brain, behaviour and immune cell populations, associated with penicillin exposure, were absent in mice that received L. rhamnosus JB-1 supplementation concurrent with the antibiotic. Our study indicates that post-natal exposure to a clinically relevant dose of antibiotic has long-term, sex dependent effects on the CNS and may have implications for the development of neuropsychiatric disorders. Importantly, we also provide further evidence that probiotic based strategies may be of use in counteracting detrimental effects of early-life antibiotics on neurodevelopment.