Analysis of adult pancreatic cells at the single cell level in vitro is useful for understanding their properties and function.However, the preparation of high quality of single cell suspension from adult pancreas is still a fundamental problem.In this study, we described a cold trypsin-EDTA digestion method for isolation of single cells from adult mouse pancreas.Through this method, a large amount of single cells (2.81 +/-0.35 x 107 per gram of tissues) was obtained, and the ratio of viable cells (91.99+/-1.59%) was higher than those got by warm collagenase digestion (65.66+/-4.96%) or warm digestion combined collagenase and trypsin-EDTA digestion (49.6+/-3.22%).