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Yak meat is rich in nutrients, but has low water-holding capacity (WHC), which affects its edible and processing quality. Therefore, control of postmortem juice loss and reduction of economic loss are crucial issues needing urgent resolution in the yak meat processing industry. Existing research suggested that changes in proteins affected the WHC of postmortem muscle, but relevant mechanism was still not entirely clear because of the complexity of protein composition. Thus, it is very important to explore the variation mechanism of postmortem WHC of yak meat by combining multiple analytical and testing methods. Proteomics research combined with bioinformatics approach was studied by comparing high-WHC (HWHC) and low-WHC (LWHC) groups. Based on the data obtained from drip loss and cooking loss, totally 18 longissimus dorsi of yak can be classified into HWHC and LWHC groups. Totally six proteins were found to be abundant differentially between HWHC and LWHC groups and identified by matrix assisted laser desorption ionizationtime of flight/time of flight (MALDI-TOF/TOF). Matched spots exhibited a three fold or more intensity difference in the meantime associated with 5% statistical significance (P<0.05). Then hydrophobicity analysis and subcellular localization prediction were used to validate the representative proteins. Results showed that differentially abundant proteins were lactate dehydrogenase (LDH), creatine kinase M-type (CKM), triosephosphate isomerase (TIM), myosin light chain (MLC), troponin T (TnT) and heat shock 27kDa (HSP27). The analysis of hydrophobicity performed that LDH, CKM and TIM had more hydrophobic regions. The results showed that the high hydrophobicity could enhance the rejection of protein and water, which might lead to the weak water retention of yak meat. In subcellular localization prediction, the differentially abundant proteins in the cells were divided into five positions. All of the differentially abundant proteins were located in the cytoplasm and nucleus. Accordingly, proteomics and bioinformatics analyses were proven to be excellent tools to quantify the changes of proteins and biological information linked to WHC, and thus helping to explain the processes behind characteristic meat quality traits in yak muscle.