To explore the therapeutic mechanism of Zhibai Dihuang Granule (ZDG) in Yin-deficiency-heat (YDH) syndrome. Methods:ITRAQ-2DLC-MS/MS technique was applied to screen the differentially expressed serum proteins between ZDG treated rats and YDH syndrome rats. The serum protein profiles were analyzed by bioinformatics tools, such as Gene Ontology (GO) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Compared with normal rats, 47 differentially expressed proteins were shown in the serum of YDH syndrome rats, including 20 up-regulated proteins and 27 down-regulated proteins. Nineteen proteins returned to normal after ZDG treatment, which mostly involved in immune reaction and metabolism. Complement C4 (C4), coagulation factor X (F10), and Src kinase-associated phosphoprotein 2 (Skap2) participated in immune response, while L-lactate dehydrogenase A chain (Ldha), Fructose-bisphosphate aldolase A (Aldoa), Arachidonate 12-lipoxygenase (Alox12), Rho GDP-dissociation inhibitor 1 (Arhgdia), and Myosin light polypeptide 6 (Myl6) mainly related to metabolic process. Conclusion:YDH syndrome is highly associated with the disturbance of immune function and metabolism, and ZDG treats YDH syndrome mostly through regulating immune activity and metabolic process.