The exposure of the human body to microgravity, conditions that occurs during space flights, causes significant changes in the cardiovascular system. Many cell types have been involved in these changes, and the endothelium seems to play a major role. In endothelial cells (EC), it has been shown that modeled low gravity impairs nitric oxide synthesis, cell adhesion, extracellular matrix composition, cytoskeleton organization, cytokines, and growth factors secretion. Nevertheless, detailed analysis of EC physiological changes induced by microgravity exposure is still lacking. Secretome analysis is one of the most promising approaches for the identification of biomarkers directly related to the physiopathological cellular state. In this study, we analyzed in details the modifications of EC secretome by using umbilical vein endothelial (HUVE) cells exposed to modeled low gravity conditions. By adopting a two-dimensional (2-D) proteomic approach, in conjunction with a technique for the compression of the dynamic range of proteins, we observed that modeled low gravity exposure of HUVE cells affected the secretion of proteins involved in the regulation of cytoskeleton assembly. Moreover, by using Luminex® suspension array systems, we found that the low gravity condition decreased in ECs the secretion of some key pro-inflammatory cytokines, including IL-1α and IL-8, and of the pro-angiogenic factor bFGF. On the contrary, microgravity increase the secretion of two chemokines (Rantes and Eotaxin), involved in leukocytes recruitment.