Functional constipation ranks among the most common disorders impacting human health, which is manifested by difficulty in defecation and a complex etiology. L-Arabinose, a pentose found naturally in fruit rinds and cereal husks, has been reported to regulate glycolipid metabolism, improve glucose homeostasis, and exhibit anti-inflammatory effects. However, the effect and precise mechanism of L-Arabinose on functional constipation remain unclear. In this study, the effect of L-Arabinose in alleviating functional constipation induced by diphenoxylate was evaluated. The model group consisted of functional constipation mice that did not receive any intervention. The positive drug group was treated with 2.0 g/kg lactulose, while the intervention group was given 0.5 g/kg, 0.75 g/kg, 1.0 g/kg, and 2.0 g/kg L-Arabinose, respectively. The data suggested that 20 days of L-Arabinose intervention could shorten the first black stool defecation time, increase fecal water content, and enhance the rate of small intestinal propulsion in mice with functional constipation induced by diphenoxylate. Additionally, L-Arabinose reversed the protein expression of functional constipation-related intestinal factors in the colon, characterized by a decrease in the expression of water channel proteins AQP3 and AQP4, as well as an increase in the expression of tight-junction proteins ZO-1, Claudin-1 and Occludin. Furthermore, L-Arabinose modulated the levels of hormones (MTL, Gas) and neurotransmitters (5-HT, VIP) related to the digestive systems of mice with constipation, resulting in elevated levels of 5-HT, MTL, and Gas and decreasing levels of VIP. Histopathological analysis also revealed that L-Arabinose intervention improved the intestinal inflammatory response. Furthermore, 16S rRNA sequencing and metabolomics of the intestinal microbiota demonstrated that L-Arabinose treatment improved both the intestinal microbiota composition and the metabolite levels. This study suggests that L-Arabinose can serve as a potential functional ingredient to promote intestinal health, enhance gastrointestinal motility and barrier function, regulate osmotic pressure, restore neurotransmitter levels, and effectively relieve functional constipation.