Constipation is directly related to the intestinal microenvironment, in which the promotion of gastrointestinal (GI) motility and improvement of gut microbiota distribution are important for alleviating symptoms. Herein, after the intervention of probiotic fermented milk (FMMIX) containing Lacticaseibacillus paracasei JY062 and Lactobacillus gasseri JM1 for 14 d in Kunming mice with loperamide-induced constipation, the results indicated that FMMIX significantly increased the secretion of serum motilin (MTL), gastrin (GAS) and 5-hydroxytryptamine (5-HT) and decreased those of peptide YY (PYY), vasoactive intestinal peptide (VIP) and nitric oxide (NO) in mice. As determined by immunohistochemical analysis, FMMIX promoted an augmentation in the quantity of Cajal interstitial cells (ICC). And mRNA and protein expression of c-kit and SCF was upregulated to facilitate intestinal motility. High-throughput sequencing and gas chromatography techniques revealed that FMMIX led to an increase in the relative abundance of beneficial bacteria (Lactobacillus, Oscillospira, Ruminococcus, Coprococcus, and Akkermansia), reduced the presence of harmful bacteria (Prevotella), and resulted in elevated levels of short-chain fatty acids (SCFAs) with a superior improvement compared with UFM. Untargeted metabolomics revealed significant upregulation of functional metabolites such as L-pipecolinic acid, DL-phenylalanine and naringenin in FMMIX, presumably playing a potential role in constipation relief. In conclusion, FMMIX had the potential to alleviate constipation symptoms in mice by improving the secretion of serum GI regulatory peptides and neurotransmitters, increasing the expression of c-kit and SCF proteins, and modulating the gut microbiota structure and SCFAs levels, and may be associated with an increase in the above functional components in FMMIX. This suggested that FMMIX could be a promising adjunctive strategy for managing constipation symptoms and could contribute to the development of functional foods aimed at improving gut health.