OBJECTIVE To investigate the mechanism of Mayuan tongbian zhitong decoction on improving slow-transmission constipation（STC）in rats by regulating AMP activated protein kinase （AMPK）/endothelial nitric oxide synthase （eNOS）signaling pathway. METHODS The rats were randomly divided into blank group ，model group ，Mayuan tongbian zhitong decoction low-dose，medium-dose and high-dose groups （6，12，18 mg/kg），with 10 rats in each group. Except for blank group ，other groups were given Compound diphenoxylate suspension to induce STC model. After modeling ，blank group and model group were given normal saline intragastrically ，and Mayuan tongbian zhitong decoction groups were given relevant medicine intragastrically ， once a day ，for consecutive 2 weeks. The number of feces and water content of feces in each group were observed before and after treatment；the carbon powder propulsion rate of rats in each group was calculated ；the pathological structure of colon in each group was observed ；the levels of nitric oxide （NO）and nitric oxide synthase （NOS）in colon tissues of rats in each group were detected；the expressions of AMPK ，eNOS，mammalian target of rapamycin （mTOR），tuberous sclerosis complex 1（Tsc-1）， Tsc-2 and eukaryotic promoter 4E binding protein （4ebp）were also detected. The active ingredients of Cannabis sativa ，Citrus aurantium and Rehmannia glutinosa were screened from Mayuan tongbian zhitong decoction. The active ingredients with high Degree value were docked with AMPK and eNOS ，to verify the interaction. RESULTS Compared with before treatment ，the number and water content of feces were increased significantly in Mayuan tongbian zhitong decoction groups （P＜0.05）. Compared with blank group ，carbon powder propulsion rate of model group was decreased significantly （P＜0.05）； colonic structure was disordered ，and a large number of inflammatory cells were seen in submucosa ；the levels of NO and NOS in colon tissue as well as the protein expressions of AMPK ，eNOS，mTOR，Tsc-1，Tsc-2 and 4ebp were increased significantly （P＜0.05）. Compared with model group ，above indexes of Mayuan tongbian zhitong decoction groups （except for NOS in low-dose group ）were reserved significantly （P＜0.05）. In the molecular docking experiment ，the active components with the highest Degree values in C. sativa ，C. aurantium and R. glutinosa were（Z）-3-（4-hydroxy-3-methoxy-phenyl）-N-[2-（4-hydroxyphenyl）ethyl] acrylamide，nobiletin and stigmasterol. The binding energies of AMPK with these three components were －5.15，－4.61 and －4.83 kJ/mol，the binding energies of eNOS with these three components were －6.11，－5.40 and －5.91 kJ/mol. The conformations of these three compounds with AMPK and eNOS were stable and their binding activities were high. CONCLUSIONS Mayuan tongbian zhitong decoction can improve the constipation symptoms and intestinal function in STC model rats ，and its specific mechanism may be related to the inhibition of AMPK/eNOS signaling pathway.