Endothelial cells constitute an interface between blood and tissue and act as a medium for active interaction between plasma and the intracellular environment for homeostasis. Aging of endothelial cells plays a significant role in the pathophysiology of age-related vascular diseases; however, precise mechanisms for senescence have not been elucidated. Proteomics allows identification of protein structures, functions, and characteristics, and can be applied to the study of aging processes. Using cultured human dermal microvascular endothelial cells and two-dimensional proteomic mapping, we studied the effects of kinetin, epigallocatechin-3-gallate, all-trans-retinoic acid, and selenium on their senescence and searched for the aging-related proteins. The treatments resulted in 68 qualitative changes and 172 quantitative changes, and we were able to identify 46 spots among them. All of the agents indicated above induced changes in the expression of moesin, rho guanosine-5'-diphosphate-dissociation inhibitor, and actin, confirmed by immunoblotting and confocal laser microscopy. As these proteins were associated with cell cycle and cytoskeleton, immunoblotting of the proteins related to cell cycle was performed. Although practical significance remains to be confirmed by in vivo research, this fundamental discovery may provide a basis for understanding the mechanism of aging and age-related diseases.