Changes in yak mitochondria by natural selection in a hypoxic environment could be utilized to understand adaptation to low-oxygen conditions. Therefore, the differences in proteome profile of skeletal muscle mitochondria from yak, dzo, and cattle were analyzed by mass spectrometry, which were then classified into 3 groups, comparing between yak and dzo, yak and cattle, and dzo and cattle. 376 unique mitochondrial proteins were identified, including 192, 191, and 281 proteins in the yak-dzo, yak-cattle, and dzo-cattle groups, respectively. NRDP1 and COQ8A were expressed at higher levels in yak and dzo compared to those in cattle, indicating higher endurance capacity of yak and dzo in a low-oxygen environment. Gene Ontology (GO) terms of biological processes were significantly enriched in oxidation-reduction process, and that of molecular functions and cellular component were enriched in oxidoreductase activity and the mitochondrion, respectively. The most significantly affected pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were Parkinson’s disease, Huntington’s disease, and oxidative phosphorylation between the yak-cattle and dzo-cattle groups; while metabolic pathways, citrate cycle, and carbon metabolism were significantly affected pathways in the yak-dzo group. ATP synthases, MTHFD1, MDH2, and SDHB were the most enriched hub proteins in the protein-protein interaction (PPI) network. These results indicated that mammals living at high altitudes could possibly possess better bioenergy metabolism than those living in the plains. The key proteins identified in the present study may be exploited as candidate proteins for understanding and fine-tuning mammalian adaptation to high altitudes.