Cardiac hypertrophy is one of the main target organ damages of essential hypertension and predicts a poor prognosis of the disease. The molecules involved in this event, especially their posttranslational modifications, remain largely unknown to date. With a combination of phosphoprotein column enrichment and two-dimensional gel electrophoresis separation, here we compared the profiling of enriched phosphoproteins from the left ventricle (LV) of spontaneously hypertensive rats (SHR) to that of age-matched Wistar Kyoto rats. As a result, 19 differential proteins were found in the hypertrophied LV of SHR. Among them, we focused on a glycolysis enzyme alpha-enolase, of which the hyper-phosphorylation was shown in the hypertrophied LV but not in non-hypertrophied atrium and right ventricle of SHR. Furthermore, the alpha-enolase hyper-phosphorylation was accompanied by decreased enzymatic activity. The further investigation based on these results would provide new clues to understand the pathological process of cardiac hypertrophy in SHR.