Extracellular signal-regulated kinase 5 (ERK5), a recently discovered mitogen-activated protein kinase (MAPK), plays a key role in the development and pathogenesis of cardiovascular disease. In order to clarify the pathophysiological significance of ERK5 in vascular remodeling, we investigated ERK5 phosphorylation in hypertrophy of human aortic smooth muscle cells (HASMCs) induced by angiotensin II (Ang II). The AT1 receptor was involved in Ang II-induced ERK5 activity. Hypertrophy was detected by the measurement of protein synthesis with [(3)H]-Leu incorporation in cultured HASMCs. Ang II rapidly induced phosphorylation of ERK5 at Thr218/Tyr220 residues in a time- and dose-dependent manner. Activation of myocyte enhancer factor-2C (MEF2C) by ERK5 was inhibited by PD98059. Transfecting HASMCs with small interfering RNA (siRNA) to silence ERK5 inhibited Ang II-induced cell hypertrophy. Thus, ERK5 phosphorylation contributes to MEF2C activation and subsequent HASMC hypertrophy induced by Ang II, for a novel molecular mechanism in cardiovascular diseases induced by Ang II.