Yak (Bos grunniens) lives at plateau area of more than 3500 altitude meter, in this case, yak still maintains normal physiological activity. Besides, yak meat is rich in protein and low in fat, which does not contain anabolic steroids. Proteomics research with bioinformatics approach combined with the established two dimensional electrophoresis (2DE) platforms was studied by comparing yak with beef cattle muscle. Aiming to illustrate the causes and pathway of different meat qualities in yak and beef cattle, establish the optimal 2DE system and analyze protein bioinformatics pathways, different lysis buffer components, isoelectric focusing procedures and staining methods were studied by using longissimus dorsi muscle of yak. Proteomic profiling by 2DE and mass spectrometry identified 19 proteins that were differentially expressed in longissimus dorsi muscle of yak and beef cattle. Then the identified proteins were analyzed by gene ontology (GO) annotations and Kyoto encyclopedia of genes and genomes (KEGG) pathway. Results showed that the optimal protein extraction methods were lysis buffer component II, progressive fast boosting program and improved coomassie blue staining method. And protein spots in yak and beef cattle were 553 and 479, respectively. Totally 19 protein spots exhibiting a teo fold or more intensity difference in the meantime associated with 5% statistical significance (P<0.05) were considered differentially abundant. The differentially abundant proteins between yak and beef cattle could be divided into three main functional categories: metabolism proteins, structure proteins and stress proteins. The method of GO annotation provided three detailed and structured terms that included cellular component, molecular function and biological process. The differentially expressed proteins in yak and beef cattle muscle were concentrated in cellular processes, carbohydrate metabolism, genetic information processing and energy metabolism pathways by KEGG pathway analysis. In conclusion, the research result demonstrated the functions of identified proteins and provided a more detailed molecular view of the processes behind meat quality in yak and beef cattle.