Although gut microbiota and early life events are likely involved in the development of irritable bowel syndrome (IBS), it remains unclear how these factors interact in the pathophysiology of IBS. In the present study, using rats subjected to maternal separation (MS) as a model of IBS, we investigated interrelationships among gut microbiota, stress susceptibility and intestinal permeability, and examined the effect of the probiotic Bifidobacterium bifidum G9-1 (BBG9-1) on those interrelationships. When compared with the controls at postnatal day 20, MS rats showed hypercorticosteronemia, enhanced intestinal permeability and changes in gut microbiota structure. All of these changes in MS rats were prevented by treatment with BBG9-1. Although the gut microbiota profile and basal serum corticosterone level did not differ between MS and control rats at postnatal day 56, MS rats showed hypersensitivity to restraint stress in terms of serum corticosterone level and fecal frequency. However, such hypersensitivity was not observed in MS rats treated with BBG9-1. These findings suggest that MS initiates the link between gut microbiota alteration and hypersensitivity to stress and that the triggering of this process can be prevented by the treatment with the probiotic BBG9-1.