Periampullary diverticulum (PAD) is frequently come upon during endoscopic retrograde cholangiopancreatography (ERCP), especially in elderly patients. However, less is known about the role of PAD in biliary cannulation difficulty. This study aims to investigate the association of PAD and difficult cannulation and evaluate the impact of different types of PAD on the cannulation success rate and adverse events. Prospectively collected data on a total of 636 patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) were divided into two groups based on the presence or absence of PAD. Besides, the patients were classified based on the PAD types into three groups. The primary outcomes were cannulation success rate, cannulation time, and ERCP-related adverse events. Further, the difficult cannulation and presence of PAD were analyzed using logistic regression models. Significant higher rates of biliary stones, cholangitis, and biliary pancreatitis were observed in the PAD group. Successful selective cannulation was achieved in 97.6% in the PAD group and 95.3% in the control group. The cannulation time was significantly longer in the presence of PAD. There was no significant difference in the rate of overall adverse events and post-ERCP pancreatic PEP. Multivariate analysis showed that type 1 PAD, biliary stones, and cholangitis were factors related to difficult cannulation. The presence of PAD did not affect the duration or success of the ERCP procedure. However, it was associated with longer cannulation time and an increase in the cannulation difficulty, especially with PAD type 1. Clinical Trial Study Registration This study is approved by Nanjing Medical University and registered at ClinicalTrial.gov PRS with ID/NCT03771547/.