Type 2 diabetes mellitus (T2DM) is a global metabolic disease with potentially life- threatening complications. Liver metabolism plays a vital role in the occurrence and development of diabetes mellitus. It has been reported that the Chinese medicinal Anemarrhenae rhizoma (AR) can relieve insulin resistance and diabetes mellitus. However, the effect on abnormal liver metabolism in diabetes mellitus is still unclear. Therefore, we extracted liver proteins of T2DM rats induced by high-fat diet (HFD) and streptozotocin (STZ), T2DM rats treated with AR extract (ARE), obesity rats (fed with HFD), and normal control rats (fed with normal diet). Then, through tandem mass tag (TMT) labeling combined with mass spectrometry (MS), we obtained the quantitative proteomic data. Bioinformatics software was used for hierarchical cluster analysis and principal component analysis of the data in each group. The volcano map for differentially expressed proteins (P < 0.05, fold change > 1.5) was plotted. It was found that the treatment group was closer to the normal control group, indicating that the quantitative proteomic data of liver tissue can reflect the therapeutic effect of ARE on T2DM rats. Key protein clusters closely related to the treatment of ARE were screened out. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the protein clusters were analyzed by David, and the result showed that AR's alleviation of abnormal fatty acid metabolism in livers of T2DM rats may be related to the regulation of the expression of key proteins Ndufa6 and Prkar2b.