Proteomic analysis is an important investigative tool used to systematically explore cellular proteins that are responsive to adverse environmental challenges. Tobacco smoking is the second major cause of death in the world. In this study, we utilized two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) technologies to explore protein changes in human aortic endothelial cells (HAECs) in response to cigarette smoke extracts (CSE). Among 389 individual proteins resolved using 2-DE, 43 had a 2- to 3-fold change in levels as measured by spot intensity and 32 had more than a 3-fold change. Sixteen of the 32 spots with sufficient amount of proteins were excised for identification by performing matrix-assisted laser desorption/ionization (MALDI)-MS analysis. Using a peptide mass fingerprinting (PMF) to search the nrNCBI database, we identified all these 16 proteins, which were either increased (n = 9) or decreased (n = 7) after CSE treatment. All these proteins have known functions, however, none have been reported to be altered after CSE treatment. The findings from our study suggest that utilizing a systemic investigative tool, such as the proteomic approach using 2-DE, may play an important role in discovering novel molecular mechanisms for cigarette smoking-induced pathological changes. Further investigation following the systemic discoveries must be further examined as they may potentially lead to new therapeutic approaches to smoking-induced diseases - a health issue affecting everyone in the world.