BACKGROUNDConsumers require fermented milk that possesses constipation‐relieving functions. To cater to the ‘natural and additive‐free’ consumption habit, this study is dedicated to developing probiotic fermented milk with constipation‐relief effects. Previously, we isolated two Lactobacillus strains, Lactobacillus MWLp‐12 and Lactobacillus MWLf‐4, from breast milk. This study evaluated the efficacy of these strains and their fermented milk in alleviating constipation in a Balb/c mouse model of constipation. The evaluation criteria included fecal water content, time for first black feces expulsion and propulsion rate of the small intestine. The mechanisms of constipation relief were investigated using gastrointestinal regulatory peptides, colonic tissue pathology, short‐chain fatty acid levels and gut microbiota analyses.RESULTSFecal water content, time for first black feces expulsion and small intestine propulsion rate indicated that both MWLp‐12 and MWLf‐4, as well as their fermented milk, could alleviate constipation in mice. Fermented milk exhibited superior effectiveness for relieving constipation compared to that of the strains alone. The results related to gastrointestinal regulatory peptides and short‐chain fatty acids suggest that the mechanisms of constipation relief by the strains and their fermented milk may involve increased levels of 5‐hydroxytryptamine and substance P in the mouse serum, higher concentrations of short‐chain fatty acids in the intestines and decreased vasoactive intestinal peptide levels in the serum.CONCLUSIONMWLp‐12, MWLf‐4 and fermented milk relieve constipation in mice. © 2024 Society of Chemical Industry.