Background and Aims <p>Difficult biliary cannulation with unintentional pancreatic duct cannulation is a challenging issue, even for experienced endoscopists. This prospective, randomized, single-center trial aims to evaluate the safety and efficacy of two advanced rescue cannulation methods in this context: transpancreatic biliary sphincterotomy and pancreatic guidewire-assisted fistulotomy.</p> Methods <p>Patients with intact papilla who were planned to undergo bile duct cannulation were screened and those who experienced difficult cannulation with at least two inadvertent pancreatic duct cannulations following unsuccessful double guidewire technique attempts were randomly assigned one of two rescue cannulation techniques: pancreatic guidewire-assisted fistulotomy and transpancreatic biliary sphincterotomy. The primary outcome was the cannulation success rate and the secondary outcome was the frequency of cannulation-related adverse events (trial registration number: IRCT20230314057717N1).</p> Results <p>Total 730 patients were screened and 194 were recruited according to the study protocol (97 in each group). Successful biliary cannulation was achieved in 93.8% (<i>n</i> = 91) of the pancreatic guidewire-assisted fistulotomy group and 81.4% (<i>n</i> = 79) of the transpancreatic biliary sphincterotomy group (<i>p</i>-value = 0.01). Multivariate analysis revealed that the transpancreatic biliary sphincterotomy technique and a normal common bile duct diameter were independently associated with unsuccessful cannulation. No significant differences were observed regarding pancreatitis and other adverse events between the two groups (<i>p</i> = 0.31).</p> Conclusions <p>In difficult biliary cannulation accompanied by inadvertent pancreatic duct cannulation, following unsuccessful double guidewire technique, pancreatic guidewire-assisted fistulotomy is superior to transpancreatic biliary sphincterotomy for biliary cannulation, with similar rates of adverse events.</p> Graphical Abstract <p></p>