Objective To investigate the impact of motilin （MTL）， neurotensin（NT） and nitric oxide synthase （NOS） on Oddi sphincter （SO） motion after cholecystectomy. Methods Oddi sphincter manometry （SOM） was performed on both Guinea pig model group （cholecystectomy） and control group （laparotomy） 12 weeks after operation. Sphincter of Oddi dysfunction （SOD） group was determined by receiver operating characteristic（ROC） curve analysis and area under curve （AUC）. Protein expression of MTL， NT and NOS in SO was also detected through integral optical density method. Meanwhile， the contents of MTL and NT in patients′ plasma of both SOD group （SO pressure＞ 40 mmHg） and control group were compared. Results AUC of 0.75 and SO pressure of more than 29.8 mmHg was determined as the standard of SOD group. MTL and NT contents （193.16±29.2 pg／mL and 104.57±19.52 pg／mL， respectively） of the model group （n=10） in plasma were significant higher than those of control group （n=11） （154.24±27.69 pg／mL and 79.65±11.24 pg／mL， respectively）， and same trend of MTL and NT protein expression in SO was detected （3 556.71±455.80 and 6 321.74±203.54 of the model group； 3 075.92±350.06 and 5 843.57±344.00 of the control group）. While NOS protein expression in model group was lower than that of the control group （2 954.21±173.54 VS 3 314.91±246.67， P＜0.05）. In clinical research， the plasma contents of MTL（350.98±24.31 pg／mL VS 319.56±23.54 pg／mL）and NT（102.39±19.56 pg／mL VS 80.45±12.35 pg／mL） in SOD group （n=15） were higher than those of the control group （n=15） （P＜0.05）. Conclusion MTL and NT contents in plasma and protein expression of MTL， NT and NOS in SO may be related to SOD. MTL and NT examinations may assist diagnosing SOD after cholecystectomy.