Although probiotics may be useful for the treatment of irritable bowel syndrome (IBS), it is unclear how probiotics play a role in colonic mucosal integrity and immunity. Here, we aimed to investigate the effect of Bifidobacterium bifidum G9-1 (BBG9-1) on colonic mucosal integrity and macrophage behavior in rats subjected to maternal separation (MS) as a model of IBS. MS pups were individually separated from their mother rats, and a proportion of the MS rats were orally administered BBG9-1. The colonic mucosal permeability was evaluated by Ussing chamber assay. The expression of tight junction proteins and cytokines and the population of CD80-positive cells was examined in the colonic tissues by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Caco2 cells were stimulated with cytokines and the transepithelial electric resistance (TEER) was measured. MS rats showed significantly higher colonic permeability and lower claudin 4 expression in the colonic epithelium relative to controls. The number of CD80-positive macrophages was significantly increased in the colonic mucosa of MS rats, accompanied by the increase of IL-6 and IFN-γ expression. BBG9-1 treatment ameliorated the increase of M1 macrophage and IL-6/IFN-γ expression in the colonic tissue of MS rats. Simultaneously, BBG9-1 treatment improved the enhanced mucosal permeability and the decreased claudin 4 expression in the colon of MS rats. IL-6 and IFN-γ, whose expression is enhanced in the colon of MS rats, significantly decreased TEER in Caco2 cells in vitro. Probiotic BBG9-1 has a preventive effect on the acceleration of colonic permeability and M1 macrophage population in maternally separated rats.