Irritable bowel syndrome-diarrhea (IBS-D) is a prevalent functional bowel disease severely impairs the quality of life of patients. Visceral hypersensitivity (VH) is the main symptom of IBS-D. Probiotics have been found to effectively alleviate VH. In this study, neonatal maternal separation (NMS) was used to induce VH in 72 rats and investigated the effects of daily gavage of 3 × 109 CFU of Lactobacillus and Bifidobacterium on alleviating the symptoms of IBS-D and behavioral interventions, as well as exploring potential mechanisms. The results demonstrated that L. acidophilus and B. breve could alleviate the symptoms of IBS-D such as VH and increased frequency of bowel movements, as well as improving non-spatial memory in NMS rats. These effects were achieved by modulating the composition of gut microbiota and metabolites (including short-chain fatty acids, deoxycholic acid and adenosine), and by regulating the expression of genes and proteins associated with the NGF/TrkA signaling pathway, tryptophan metabolism, and inflammatory factors in the hippocampus. This finding suggests that L. acidophilus and B. breve have promising potential for the alleviation of IBS.