Constipation is a prevalent gastrointestinal (GI) problem affecting a large number of individuals. This study aimed to investigate peristalsis-promoting potential characteristics of Ligilactobacillus acidipiscis YJ5 and the underlying molecular mechanism. The study demonstrated the relieving effect of L. acidipiscis YJ5 on constipation in both zebrafish and mouse models. L. acidipiscis YJ5 intervention significantly increased intestinal peristalsis by reducing the peak time and increasing the fluorescence disappearance rate in the zebrafish model. In the mouse model, the symptoms of constipation relief induced by L. acidipiscis YJ5 included a shortened first black stool time, an increased number of defecation particles, an accelerated propulsion rate of the small intestine, and an increase in fecal water content. L. acidipiscis YJ5 was found to reduce the expression of colonic aquaporins to normalize the colonic water transport system of constipated mice. Additionally, L. acidipiscis YJ5 reversed loperamide-induced morphological damage in the ileum and colon and increased the colonic mucosal barrier. The results of the 16S rRNA gene analysis indicated that L. acidipiscis YJ5 could reverse the structure of gut microbiota to a near-normal group, including levels of β-diversity, phylum, family, and genus. Furthermore, the fermentation supernatant of L. acidipiscis YJ5 was shown to relieve constipation, and metabolomics analysis revealed that these positive effects were related to its metabolites like malic acid and heliangin.