Objective To investigate the effect of nitroglycerin on preventing post-endoscopic retrograde cholangiopancreatograph (ERCP) pancreatitis (PEP) and hyperamylasemia. Methods One hundred patients diagnosed as common bile duct stones by CT or MRI and planned to undergo ERCP, EST and stones removal under endoscopy were selected from January to December 2008 in Shandong Jiaotong Hospital. These patients were randomly divided into 2 groups: nitroglycerin group (n=50), in which 0.5 mg nitroglycerin was given sublingually in 5-10 min before ERCP; control group (n=50), in which no nitroglycerin was given. The levels of serum amylase of all the patients before ERCP and at 3 h, 24 h after ERCP were detected and the incidence of hyperamylasemia and PEP were also observed. Results The level of serum amylase between 2 groups before ERCP was not significantly different (P＞0.05). The levels of serum amylase at 3 h and 24 h after ERCP were significantly higher than that before ERCP in 2 groups. The level of serum amylase in nitroglycerin group were respectively lower than that in control group at 3 h and 24 h after ERCP 〔3 h: (108.88±152.07) U/L vs. (196.30±244.41) U/L; 24 h: (97.02±113.38) U/L vs. (234.22±406.05) U/L〕， P＜0.05. The incidence of hyperamylasemia (12.00%, 6/50) and PEP (2.00%, 1/50) in nitroglycerin group was respectively significantly lower than that in control group (hyperamylasemia: 30.00%, 15/50; PEP: 14.00%, 7/50）， P＜0.05. Conclusion Sublingual nitroglycerin can decrease the level of serum amylase and prevent PEP and hyperamylasemia.