Advanced glycation end products (AGEs) accumulated in different pathological conditions have the potent capacity to alter cellular properties which include the endothelial structural and functional regulations. The disruption of endothelial barrier integrity may contribute to AGE-induced microangiopathy and macrovasculopathy. Previous studies have shown that AGEs induced the rearrangement of actin and subsequent hyperpermeability in endothelial cells (ECs). But the mechanisms involved in this AGE-evoked EC malfunction are not well understood. This study is to directly evaluate the involvement of moesin phosphorylation in AGE-induced alteration and the effects of RhoA and p38 MAPK pathways on this process. Using immortalized human dermal microvascular endothelial cells (HMVECs), we first confirmed that ERM protein moesin is required in AGE-induced F-actin rearrangement and hyperpermeability responses in ECs by knockdown of moesin protein expression with siRNA. we then detected AGE-induced moesin phosphorylation by Western blotting. The mechanisms involved in moesin phosphorylation were analyzed by blocking the AGE-receptor binding and inhibiting Rho and MAPK pathways. AGE-treated HMVECs exhibited a time and dose-dependent increases in threonine 558 phosphorylation of moesin. The increased moesin phosphorylation was attenuated by pre-administrations of AGEs receptor antibody, ROCK or p38 inhibitor. Suppression of p38 activation via expression of dominant-negative mutants with Ad.MKK6b or Ad.p38 also decreased moesin phosphorylation. Activation of p38 pathway by transfecting HMVECs with adenoviral construct of dominant active MKK6b resulted in moesin phosphorylation. The results suggest a critical role of moesin phosphorylation in AGE-induced EC functional and morphological regulations. Activations of ROCK and p38 pathways are required in moesin phosphorylation.