Despite the worldwide occurrence of coronary atherosclerotic heart disease (CAHD), the pathogenic mechanisms underlying this disease remain largely unknown. In this study, the experimental model of atherosclerosis in rat (CAHD rat) was established by the injection of vitamin D3 associated with high fat diet for 6 weeks. By using the proteomic approach, we comparatively analyzed the proteome of the control and CAHD rat left ventricular myocardial tissues. We reproducibly separated over 2500 polypeptides by using two-dimensional electrophoresis (2-DE) at pH range of 3-11. Among these proteins, 26 proteins with large amount were identified using micro high performance liquid chromatography mass spectrometer/mass spectrometer (micro-HPLC-MS/MS). Using PDQUEST software to process the 2-DE gel images, 38 protein spots that significantly altered in CAHD were detected. Of these, 12 proteins were identified with high confidence by using 2-DE and matrix-associated laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The identification of protein alterations specify to CAHD would clarify the pathogenetic mechanisms involved in the disease and might be of prognostic and therapeutic benefit.