Continuous hemodiafiltration (CHDF) has recently been used for treatment of severe acute pancreatitis. CHDF is capable of eliminating small molecules from blood, but whether trypsin can be eliminated by CHDF is not clear. In this study, elimination of trypsin-like enzyme activity (TLE) and cationic trypsin-like immunoreactivity (TLI) using low-volume CHDF was examined at the first CHDF session in eight patients with severe acute pancreatitis. CHDF was performed with a polysulfone hemofilter (membrane area, 0.7 m2) and nafamostat mesilate, a protease inhibitor and anticoagulant, at a blood flow rate of 100 ml/min and a filtration and dialysis flow rate of 10 ml/min each. Before beginning CHDF, plasma TLE was 3.41 ± 2.86 nmol/(ml·min), and TLI was 5,900 ± 9,008 ng/ml. The average plasma clearances of TLE and TLI achieved by the circuit during the 12-hour therapy were 56.7 ± 4.9 ml/min and 8.0 ± 7.2 ml/min, respectively. The average plasma clearance of TLI into the waste fluid was 2.4 ± 1.6 ml/min whereas TLE was below the measurable sensitivity. The plasma concentration of TLE and TLI remained unchanged. These results indicate that low-volume CHDF using nafamostat mesilate as an anticoagulant can increase trypsin plasma clearance. However, low-volume CHDF is not effective to eliminate the plasma trypsin concentration.