To investigate the laxative mechanism of Zhizhu Yuli Formula on constipation mice with spleen-intestine qi dysfunction in ascending and descending based on the theory of qi movement，providing experimental evidence for its clinical application.Methods:Male Kunming mice were randomly divided into a blank group，a model group，a positive drug group，and a Zhizhu Yuli Formula group，with 10 mice in each group.The positive drug group received Maren soft capsule suspension at 0.1 g/kg，the Zhizhu Yuli Formula group received Zhizhu Yuli Formula solution at 0.483 g/kg，and the blank and model groups received an equivalent volume of solvent.After 14 days of continuous administration，the mice were given the modeling drug loperamide hydrochloride(4 mg/kg) via gavage to establish a constipation model with spleen-intestine qi dysfunction in ascending and descending.The small intestine ink propulsion rate，the time to the first black stool，the number and weight of black stools within 6 hours，and the levels of serum vasoactive intestinal peptide(VIP)，5-hydroxytryptamine(5-HT)，substance P(SP)，nitric oxide(NO)，motilin(MTL)，cholecystokinin(CCK)，peptide YY(PYY)，and calcitonin gene-related peptide(CGRP) were measured.Results:Compared with the model group，the Zhizhu Yuli Formula group showed a significant increase in small intestine ink propulsion rate(P<0.01)，a significant reduction in the time to the first stool(P<0.01)，and a significant increase in the number，weight，and water content of fecal pellets within 6 hours(P<0.01).Moreover，the serum levels of VIP，NO，CCK，and PYY were significantly reduced(P<0.01)，while the levels of SP，CGRP，and MTL were significantly increased(P<0.01).Conclusion:Zhizhu Yuli Formula has a significant laxative effect，and its mechanism may be related to regulating the spleen-intestine qi dysfunction in ascending and descending，restoring the dynamic balance of gastrointestinal neurotransmitters and hormones，regulating qi movement，and maintaining smooth qi flow.