Abstract: This experiment was conducted to isolate urease active protein from cow rumen. Rumen contents were collected from Holstein cows via fistula. Cell free rumen fluid and ruminal cellular protein fluid were collected by centrifugation and ultrasonication. After precipitated by the addition of solid (NH4)2SO4 to 85% saturation and dialyzed, the fluid was applied to a HiTrap Capto Q ion exchange column. The fractions with urease activities were pooled, and concentrated by lyophilization. Then urease was separated by native-PAGE, and identified by modified Fishbein staining. The urease strap was excised from the gels, digested by trypsin and then analyzed by LCMS/MS. Searches of the peptide mass fingerprint data against NCBI databases were performed with the SEQUEST software. The results showed that higher activity urease was isolated and purified from cell free rumen fluid and ruminal cellular protein fluid. After staining, one brownish red strap containing urease from ruminal cellular protein fluid was observed. Three kinds of urease from Streptococcus thermophilus, Streptococcus salivarius and Bacillus halodurans were detected by mass spectrum. These results demonstrated the feasibility of isolating urease protein directly from rumen mixture without microorganism cultivation and this strategy could be expected to facilitate the research on uncultured microorganisms in rumen.