Breast Cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC-associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot study, using one-dimensional polyacrylamide gel electrophoresis (1D-PAGE)-based mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)-based MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her mil. Statistically different gel spots were picked for protein digestion followed by nanoLC-MS/MS analysis. The upregulated proteins in BC versus control are alpha-amylase, gelsolin isoform a precursor, alpha-2-glycoprotein 1 zinc isoform CRA_b partial, apoptosis-inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease.