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Lycopene, a red pigmented carotenoid present in many fruits and vegetables such as tomatoes, has been associated with the reduced risk of breast cancer. This study sought to identify proteins modulated by lycopene during cell proliferation of the breast cancer cell line MCF-7 to gain an understanding into its mechanism of action. MCF-7 breast cancer cells and MCF-10 normal breast cells were treated with 0, 2, 4, 6, 8, and 10 μM of lycopene for 72 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium reduction assay was used to measure cell proliferation and two-dimensional fluorescence difference gel electrophoresis to assess the changes in protein expression, which were identified using MALDI-ToF/ToF (matrix-assisted laser desorption ionization tandem time-of-flight) and Mascot database search. MTT and cell proliferation assays showed that lycopene selectively inhibited the growth of MCF-7 but not MCF-10 cells. Difference gel electrophoresis analysis revealed that proteins in the MCF-7 cells respond differently to lycopene compared with the MCF-10 cells. Lycopene altered the expression levels of proteins such as Cytokeratin 8/18 (CK8/18), CK19 and their post translational status. We have shown that lycopene inhibits cell proliferation in MCF-7 human breast cancer cells but not in the MCF-10 mammary epithelial cells. Lycopene was shown to modulate cell cycle proteins such as beta tubulin, CK8/18, CK19 and heat shock proteins. Copyright © 2012 John Wiley & Sons, Ltd.