Chromium (Cr) is a highly toxic, common heavy metal used in industrial production. There are two types of Cr in nature: hexavalent chromium (Cr(VI)) and chromium trichloride (Cr(III)). Cr(III) is involved in the metabolism of sugars and lipids, whereas Cr(VI) is absorbed through the respiratory tract and skin and generates free radicals that result in secondary toxicity. Cr(VI) leads to cancer in the occupational population and is therefore recognized as a human carcinogen by the International Agency for Research on Cancer. The specific mechanism underlying Cr-induced carcinogenesis is complex. In this study, two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry-based techniques were performed to analyze differentially expressed proteins between Beas-2B human bronchial epithelial cells and Cr(VI)-transformed Beas-2B cells. Many differentially expressed proteins were identified in the cells after malignant transformation, including serine/threonine kinase 11, endothelial nitric oxide synthase 3, apolipoprotein A1, vinculin, and lamin A/C. These proteins are involved in many signaling and metabolic pathways, including apoptosis, autophagy, the PI3K/Akt signaling pathway, focal adhesion, cell motility, and actin cytoskeleton rearrangement.