It is thought that accurate risk assessment and early diagnosis of breast cancer (BC) can help reduce cancer-related mortality. Proteomics analysis of breast milk may provide biomarkers of risk and occult disease. Our group works on the analysis of human milk samples from women with BC and controls to investigate alterations in protein patterns of milk that could be related to BC. In the current study, we used mass spectrometry (MS)-based proteomics analysis of 12 milk samples from donors with BC and matched controls. Specifically, we used one-dimensional (1D)-polyacrylamide gel electrophoresis (PAGE) coupled with nanoliquid chromatography tandem MS (nanoLC-MS/MS), followed by bioinformatics analysis. We confirmed the dysregulation of several proteins identified previously in a different set of milk samples. We also identified additional dysregulations in milk proteins shown to play a role in cancer development, such as Lactadherin isoform A, O-linked N-acetylglucosamine (GlcNAc) transferase, galactosyltransferase, recoverin, perilipin-3 isoform 1, histone-lysine methyltransferase, or clathrin heavy chain. Our results expand our current understanding of using milk as a biological fluid for identification of BC-related dysregulated proteins. Overall, our results also indicate that milk has the potential to be used for BC biomarker discovery, early detection and risk assessment in young, reproductively active women.