Multidrug resistance (MDR) is a major obstacle to successful cancer treatment. To understand the mechanism of MDR, many cancer cell lines have been established, and various mechanisms of resistance, such as ATP-binding cassette (ABC) transporter-mediated drug efflux, have been discovered. Previously, a MDR cell line MCF7/AdVp3000 was selected from breast cancer cell line MCF7 against Adriamycin, and overexpression of ABCG2 was thought to cause MDR in this derivative cell line. However, ectopic overexpression of ABCG2 in MCF7 cells could not explain the extremely high drug resistance level of the selected MCF7/AdVp3000 cells. We hypothesized that MCF7/AdVp3000 cells must have other resistance mechanisms selected by Adriamycin. To test this hypothesis, we compared the global protein profiles between MCF7 and MCF7/AdVp3000 cells. Following two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, 17 protein spots with differential levels between the two cell lines were identified. Although 14-3-3sigma, keratin 18, keratin 19, ATP synthase beta, protein disulfide isomerase, heat shock protein 27, cathepsin D, triose-phosphate isomerase, peroxiredoxin 6, and electron transfer flavoprotein were increased, nm23/H1, peroxiredoxin 2, nucleophosmin 1/B23, and inorganic pyrophosphatase were decreased in MCF7/AdVp3000 cells. The differential levels of these proteins were validated using Western blot. Furthermore, functional validation showed that the elevated 14-3-3sigma expression contributes considerably to the observed drug resistance in MCF7/AdVp3000 cells. We, thus, conclude that these proteins likely contribute to the resistance selected in the MCF7/AdVp3000 cells, and their altered expression in tumors may cause clinical resistance to chemotherapy.