In this study, in order to evaluate the alleviating effect of Bifidobacterium bifidum on constipation, a mouse model of constipation was established by gavaging mice with loperamide. After four weeks of intervention with B. bifidum CCFM1167 alone or in combination with B. longum subsp. longum CCFM1113 or Lacticaseibacillus rhamnosus CCFM1060, constipation-related indicators, intestinal barrier function, inflammatory cytokines, and gut microbiota were evaluated. The results demonstrated that both single and combined strains alleviated constipation, but the underlying mechanisms were different. CCFM1167 alleviated constipation primarily by promoting intestinal mucus secretion. In contrast, both probiotic combinations relieved constipation by repairing the intestinal mechanical and immune barriers. This study elucidates the potential mechanisms of action of combined probiotics in alleviating constipation, providing a basis for the formulation of probiotic combinations for constipation relief.