Certain probiotics have beneficial effects on the function of the central nervous system through modulation of the gut-brain axis. Here, we describe a dynamic landscape of the peptidome across multiple brain regions, modulated by oral administration of different probiotic species over various times. The spatiotemporal and strain-specific changes of the brain peptidome correlated with the composition of the gut microbiome. The hippocampus exhibited the most sensitive response to probiotic treatment. The administration of heat-killed probiotics altered the hippocampus peptidome but did not substantially change the gut microbiome. We developed a literatu-remining algorithm to link the neuropeptides altered by probiotics with potential functional roles. We validated the probiotic-regulated role of corticotropin-releasing hormone by monitoring the hypothalamic-pituitary-adrenal axis, the prenatal stress-induced hyperactivity of which was attenuated by probiotics treatment. Our findings provide evidence for modulation of the brain peptidome by probiotics and provide a resource for further studies of the gut-brain axis and probiotic therapies.