It is yet not clear whether differences in the contrast medium may affect post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP). The aim of this study was to evaluate the rate of PEP between high osmolality contrast medium (HOCM) and iso osmolar medium (IOCM) in a prospective, randomized, controlled trial. Patients who were scheduled to undergo a diagnostic or therapeutic procedure under ERCP guidance were included. Patients were excluded from this study if they (1) were < 20 years old; (2) had acute or active pancreatitis; (3) had previously undergone ERCP; (4) had surgical altered upper gastrointestinal tract anatomy; or (5) refused to be involved in this study. Seventy-five patients were randomly assigned to the IOCM group, and 71 patients were assigned to the HOCM group. PEP occurred in 27 patients (18.5%). Although there were no significant differences, the severe grade of PEP was more frequent in the HOCM group than in the IOCM group (p = 0.08). The results of the multivariate analyses for PEP were not identified as independent risk factors. Also, independent risk factors of hyperamylasemia were performing pre-cut. In subgroup analyses, HOCM was an independent risk factor for hyperamylasemia. In conclusion, HOCM may play a role in the development of PEP and in PEP severity grade.