A Fosmid library of uncultured microorganism from Hu sheep rumen was constructed to explore the potential of the ruminants’ gut ecosystem， and the library storage capacity, stability and the functionality was also analyzed. A total of 12 704 clones were acquired from the Fosmid library. Restriction analysis by Xho Ⅰ and BamH Ⅰ revealed a high level of diversity of the cloned DNA fragments. The insert size of clone ranged from 17 to 55 kb, with average insert size of 30.9 kb, which mostly distributed between 36～40 kb and 26～30 kb. The capacity of this Fosmid library was 393 Mb. It was demonstrated by restriction analysis with Hind Ⅲ that the Fosmid library clones cultured for 100 generations still showed good stability. With the Congo red stain analysis, 18 clones had been acquired with the activity of xylanase. It was suggested that the constructed Fosmid library had good storage capacity, stability and the functionality, and could be used for further research on exploration of enzyme resource.［Chinese Journal of Animal Nutrition, 2010，22（2）:341-345］