Probiotics have received wide attention as a potential way to alleviate gastrointestinal (GI) motility disorders. Herein, we investigated the effects of Lacticaseibacillus paracasei JY062, Lactobacillus gasseri JM1, and the probiotic combination at 5 × 109 CFU/mL on mice induced by loperamide and explored the possible underlying mechanisms in GI motility disorder. After two weeks of probiotic intervention, the results indicated that the probiotic combination alleviated GI motility disorder better. It increased the secretion of excitatory GI regulators motilin, gastrin, and 5-hydroxytryptamine (5-HT) and decreased the secretion of the inhibitory GI regulators peptide YY and nitric oxide (NO), except vasoactive intestinal peptide. 5-HT and NO were related to the mRNA expression of 5-HT4 receptor and nitric oxide synthase, respectively. The intervention of probiotic combination also increased the number of interstitial cells of Cajal and the expression of SCF/c-kit protein. In addition, it also increased the abundance of beneficial bacteria (Lactobacillus, Rikenellaceae, and Clostridiaceae_Clostridium) and improved the contents of short-chain fatty acids in cecum contents of mice. In conclusion, the probiotic combination of L. paracasei JY062 and L. gasseri JM1 has the potential to alleviate GI motility disorders by balancing intestinal homeostasis.