Background and Study Aims. The usefulness of wire-guided cannulation for avoiding hyperamylasemia and pancreatitis following endoscopic retrograde cholangiopancreatography (ERCP) is conflicting, and therefore we designed this study to determine whether wire-guided cannulation reduces the rate of post-ERCP hyperamylasemia and pancreatitis and compare its efficacy to conventional method. Patients and Methods. Seven hundred and forty-eight patients with hepatobiliary diseases consecutively underwent diagnostic or therapeutic ERCP at the unit of Taleghani referral hospital in Tehran. Among them, 546 patients were eligible for wire-guided cannulation and underwent this procedure and others underwent sphincterotome biliary cannulation using contrast injection as the conventional method. Results. Patients in the two groups were comparable in terms of gender and age. Successful biliary cannulation was achieved similary in the guidewire and conventional group (89.2% versus 86.4%) that in 5.4% and 14.1% of them it was difficultly performed, respectively (P = 0.003). The main pancreatic duct was more visualized in 99.0% of patients in conventional group in comparison with 79.0% in another group (P < 0.001). Multivariate regression analysis showed that wire-guided cannulation had a protective role for post-ERCP hyperamylasemia (OR: 0.336, 95% CI: 0.181-0.623, P < 0.001). However, there were no significant differences between the two groups in rates of other procedure-related complications, such as, pancreatitis, bleeding, and perforation. Conclusion. The use of guidewire cannulation in comparison with conventional method can be accompanied with lower post-ERCP hyperamylasemia, and therefore selection of this cannulation technique especially in high-risk group is recommended.