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Background and Aim: Wire-guided cannulation (WGC) might increase the biliary cannulation rate and decrease the risk of post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP). We assessed the learning curve for WGC in therapeutic biliary ERCP (study 1) and compared WGC and conventional contrast-assisted cannulation (CC) by a matched case-control study (study 2). Methods: Prospectively collected data of 500 therapeutic biliary ERCP cases (250 consecutive cases of WGC and 250 matched controls of CC) were retrospectively studied. Rate and time of biliary cannulation, total procedure time, PEP, and hyperamylasemia were analyzed. Results: In study 1, biliary cannulation by WGC was successful in 96% of the first 50 cases, with a median time to cannulation of 3 min. Rates of hyperamylasemia were within 10% after 100 WGC. In study 2, there were no significant differences in the overall cannulation rate and PEP between WGC and CC, but the total procedure time was shorter in WGC (30 vs 35 min, P = 0.059). Rates of hyperamylasemia and the change in serum amylase levels was lower (9% vs 14%, P = 0.069, and + 62.8 U/L vs+ 169.5 U/L, P = 0.043) in WGC, which was more prominent in experienced endoscopists (9% vs 17%, P = 0.025, and + 68.9 U/L vs+ 229.3 U/L, P = 0.014). Conclusions: The introduction of WGC was effective in the first 50 cases and did not increase the rate of PEP in biliary therapeutic ERCP.