In this study, we systemically evaluated the effects of oral administration of L. plantarum GUANKE on constipation symptoms, water channel protein expression, and the SCF/c-kit pathway in colon tissues via immunohistochemistry and the quantification of neurotransmitters and hormones in serum, short-chain fatty acid abundance in cecum content, and fecal gut microbiota using a diphenoxylate-induced constipation mouse model, which may provide evidence for the utilization of probiotics in preventing constipation. Here, KIT is linked to constipation disorder.