Abstract: A2 milk, as an emerging dairy product, has gained increasing market recognition; however, studies on its fermented products remain limited. Our previous work demonstrated that probiotic strains derived from human breast milk, when used to ferment conventional milk, exert significant anti-constipation effects. Building on this foundation, the present study systematically compared the efficacy of two types of fermented A2 milk in alleviating constipation: one produced using a commercial starter culture alone (CA2), and the other produced using a combination of the commercial starter culture supplemented with two human milk-derived probiotic strains, Lactiplantibacillus plantarum MWLp-12 and Limosilactobacillus fermentum MWLf-4 (A2). A loperamide-induced constipation mouse model was established to comprehensively evaluate defecation-related parameters, including fecal water content, small intestinal transit rate, and time to first black stool. In addition, serum levels of gastrointestinal regulatory peptides—5-hydroxytryptamine, motilin, substance P, somatostatin, and vasoactive intestinal peptide—as well as fecal short-chain fatty acids (SCFAs) and gut microbiota composition were analyzed to assess the regulatory effects of the interventions. The results indicated that both fermented A2 milk products significantly increased fecal water content and small intestinal transit rate, and reduced the time to first black stool in constipated mice (P<0.05), with the A2 group demonstrating greater efficacy than the CA2 group (P<0.05). In terms of serum gastrointestinal regulatory peptide levels, both fermented products elevated the levels of excitatory peptides, including 5-hydroxytryptamine, motilin, and substance P, while decreasing the levels of inhibitory peptides such as somatostatin and vasoactive intestinal peptide. Notably, the A2 group exhibited significantly stronger modulatory effects on motilin and substance P compared to the CA2 group (P<0.05). Furthermore, both fermented A2 milk products increased fecal short-chain fatty acid concentrations, including acetic, propionic, and butyric acids, and improved constipation-induced gut microbiota dysbiosis by significantly enriching beneficial bacteria, such as Lactobacillus and Lachnospiraceae, while reducing the abundance of opportunistic pathogens. Compared to the CA2 group, the A2 group significantly increased the relative abundance of Muribaculaceae and Bacteroidaceae, while decreasing the abundance of harmful bacteria like Desulfovibrionaceae (P<0.05). Histopathological observations of colon tissues revealed that both interventions mitigated constipation-induced damage to colonic crypt structures. In conclusion, fermented A2 milk effectively alleviates constipation, with the product supplemented with MWLp-12 and MWLf-4 demonstrating superior efficacy. The underlying mechanisms involve the regulation of serum gastrointestinal peptide levels, enhancement of intestinal SCFAs production, and improvement of gut microbiota composition. This study provides a theoretical foundation for the development of fermented A2 milk with constipation-alleviating functionality.