Background: Endoscopic retrograde cholangiopancreatography (ERCP) is an important therapeutic tool for many biliary diseases. Selective biliary cannulation is the first step of the treatment process. Needle knife fistulotomy (NKF) and conventional precut sphincterotomy (CPS) are widely used in difficult biliary cannulation. However, there are different results in their effectiveness and safety. This study aimed to compare both methods regarding cannulation success and adverse event profile.Materials and Methods: All eligible consecutive patients with naive papillae who underwent biliary ERCP by a single experienced endoscopist over a 3-year period were included retrospectively. The standard cannulation method with a guidewire-loaded sphincterotome was initially used for biliary cannulation in all patients. Cannulation was accepted as difficult in the case of failure of standard cannulation within 5 minutes or despite 5 attempts or insertion of the guidewire to the pancreatic duct 5 times. Three modalities in patients with difficult biliary cannulation were employed according to the structure and configuration of the papillae in addition to unintentional pancreatic cannulation: (1) NKF, (2) CPS, (3) Double guidewire technique or guidewire orientation/ precut following pancreatic stenting. Latter was excluded to enable direct comparison between NKF and CPS groups.Results: A total of 644 patients were recruited. Analyses were performed with 541 patients after the exclusion of 103 patients. Mean (SD) age was 60.4 (18.2) years, and 257 (47.5%) patients were male. While standard cannulation was successful in 366 (67.6%), difficult biliary cannulation was observed in 175 (32.4%) patients. NKF was performed in 101 (57.7%) patients, and cannulation success was 100% in the first ERCP session. In contrast, CPS was performed in 74 (42.3%) patients with a lower cannulation success rate (79.7%) than NKF (P < 0.001). Post-ERCP pancreatitis rate was higher in CPS (9.5%) than NKF group (3.0%, P = 0.063). Bleeding and cholangitis were similar in both groups.Conclusions: In patients with difficult biliary cannulation with appropriate papillary structure and configuration, NKF should be used as the first choice in experienced hands because of high biliary cannulation success and low Post-ERCP pancreatitis risk.