The adverse effects of weightlessness on the human cardiovascular system greatly hinder the process of long-term and long-distance space exploration. Succinylation is an important type of protein post-translational modification. However, whether succinylation modification is able to play a role in altered vascular endothelial cell function under microgravity or simulated microgravity has not been reported. This study aims to investigate the quantitative global proteome and the changes in lysine succinylation in related proteins, seeking to facilitate a better understanding of the protein post-translational modification in cardiovascular deconditioning under microgravity. LC-MS/MS combined with bioinformatics analysis were used to quantitatively detect the perspectives at the global protein level. Immunoprecipitation and Western blot analysis were conducted to further verify the alterations of related proteins and lysine succinylation. A total of 132 differentially expressed proteins and 164 differentially expressed lysine succinylation sites were identified in human umbilical vein endothelial cells (HUVECs). Bioinformatics analysis indicates that lysine succinylation may play a potential role in energy metabolism. In addition, desuccinylase SIRT5 was downregulated and regulated succinylation modification levels of HUVECs under simulated microgravity. Notably, the overexpression of SIRT5 effectively protected HUVECs from apoptosis induced by simulated microgravity. And the succinylation of Lys396 in ERO1A was significantly increased in HUVECs under simulated microgravity. Mechanistically, the knockdown of SIRT5 was found to induce the apoptosis of HUVECs through the succinylation of Lys396 in ERO1A. These results can provide new ideas for elucidating the molecular mechanism of cardiovascular dysfunction in microgravity environments, and provide key molecular targets for scientific protective measures against microgravity in space.