To explore the different expression of ethyl acetate extract of Cremastra appendiculata (Cr Ap) against 4T1 breast cancer for screening its new revelation,tandem mass tag (TMT)-based quantitative proteomics was employed to screen the differential expression proteins treated by Cr Ap in breast cancer tissue．Multiple Bioinformatics databases were used to analyze the differentially expressed proteins．QPCR was used to detect Ly6G,S100a8,S100a9,Tmsb4x and GADPH mRNA expression in cancer tissues.Totally 16 differentially expressed proteins were identified.Among them,15 proteins were up-regulated and 1 protein was down-regulated.Expression of thymosin β-4 mRNA was down-regulated significantly,but Toll 4 receptor binding protein,Toll-like receptor binding protein,S100-a8/a9 and mast cell expression membrane protein 1 were up-regulated,which may be related to pro-apoptotic effect and immune regulation of Cr Ap against breast cancer.KEGG enrichment pathway analysis of the differentially expressed proteins were enriched to transcriptional misregulation and IL-17 signaling pathway in cancer signaling pathway.Cr Ap significantly increased the expression of Ly6G,S100a8,and S100a9 genes in breast cancer tissues,and reduced expression of Tmsb4x genes.The immune-related differentially expressed proteins were effectively identified by TMT-based quantitative proteomics between Cr Ap and breast cancer control,and the immunoregulatory effect of Cr Ap against 4T1 breast cancer would be expected to become a new research direction.