[Background] Lipase is a special ester bond hydrolase and widely used in industries. Microorganisms are the main source of lipase. Many microorganisms in the rumen are of great diversity. Although it has been reported about the production of cellulase by rumen microorganisms, there is no report about lipase produced by rumen microorganisms. [Objective] The purpose of this study was to isolate and screen lipase produced by microorganisms from the rumen of yak, and to identify the strains and characterize the enzymes. [Methods] Olive oil was used as the sole carbon source in neutral red fat medium for preliminary screening, then the obtained strains were rescreened by the improved copper soap-photometric. Finally, the rescreened strains were identified by morphological observation, physiological and biochemical experiments and 16S rRNA gene sequence analysis. The lipases produced by the isolated microorganisms were tested by temperature, pH, metal ions, organic solvents and surfactant, accordingly we had found the optimum conditions. [Results] Six strains with high enzyme activity were obtained. Three were Serratia liquefaciens, two were Geotrichum candidum, and one was Mucor circinelloides. Enzymatic properties showed that the optimal reaction temperature of three lipases (Serratia liquefaciens, Geotrichum candidum and Mucor circinelloides lipase) was 45, 35 and 40 °C, and the optimal reaction pH of them was 8.0, 7.0 and 7.0 respectively. The activities of these lipases were stimulated by Ca2+ and Mg2+ ions and inhibited by Zn2+ ions. Lipases were inactivated by EDTA and SDS. These lipases tolerated glycerol, while the lipase of Mucor circinelloides showed a better tolerance to methanol, ethanol and acetone than others. [Conclusion] We isolated three lipases produced by microorganisms from the rumen of yak. This study showed that rumen microorganisms are of high value in lipase research.