Abstract： Objective To validate the establishment of a novel method for isolation of intestinal epithelium stem cells using density gradient centrifugation.Methods The model of severe small intestinal mucosa damage in mice was established by intraperitoneal injection of high-dose 5-fluorouracil(5-FU). Then the damaged intestinal mucosa was digested and prepared into single cell suspension.Percoll gradient centrifugation was used to separate the cells into subsets based on their sizes. The morphological characteristics of these cell subsets were studied with HE staining, and the musashi-1 expression was detected by immunocytochemistry and RT-PCR.Results Most of the cells were located in the 50% and 70% layers of Percoll separating medium.The cells in the 50% layer displayed stem cell-like morphology. Immunocytochemistry showed positive musashi-1 expression in about 93% of these cells, and RT-PCR also revealed high level of musashi -1 expression.Conclusion A simple and effective method for isolation of viable intestinal stem cells was established in this study.