Brain metastases are a devastating consequence of cancer and currently there are no specific biomarkers or therapeutic targets for risk prediction, diagnosis, and treatment. Here the proteome of the brain metastatic breast cancer cell line 231-BR has been compared with that of the parental cell line MDA-MB-231, which is also metastatic but has no organ selectivity. Using SILAC and nanoLC-MS/MS, 1957 proteins were identified in reciprocal labeling experiments and 1584 were quantified in the two cell lines. A total of 152 proteins were confidently determined to be up- or down-regulated by more than twofold in 231-BR. Of note, 112/152 proteins were decreased as compared with only 40/152 that were increased, suggesting that down-regulation of specific proteins is an important part of the mechanism underlying the ability of breast cancer cells to metastasize to the brain. When matched against transcriptomic data, 43% of individual protein changes were associated with corresponding changes in mRNA, indicating that the transcript level is a limited predictor of protein level. In addition, differential miRNA analyses showed that most miRNA changes in 231-BR were up- (36/45) as compared with down-regulations (9/45). Pathway analysis revealed that proteome changes were mostly related to cell signaling and cell cycle, metabolism and extracellular matrix remodeling. The major protein changes in 231-BR were confirmed by parallel reaction monitoring mass spectrometry and consisted in increases (by more than fivefold) in the matrix metalloproteinase-1, ephrin-B1, stomatin, myc target-1, and decreases (by more than 10-fold) in transglutaminase-2, the S100 calcium-binding protein A4, and l-plastin. The clinicopathological significance of these major proteomic changes to predict the occurrence of brain metastases, and their potential value as therapeutic targets, warrants further investigation.