Aim To explore the effect of succinate/G protein coupled receptor 91 (GPR91) on mitochondria in vascular endothelial cells and its regulatory mechanisms. Methods Transmission electron microscopy, Western blot and fluorescence microscopy were used to observe the effects of succinate analogues diethyl succinate (DS), GPR91 agonist and inhibitor on the mitochondrial morphology, cristae, cristate homeostasis related proteins reactive oxygen species (ROS) content, Ca²＋ concentration, mitochondrial membrane potential, the expression of dihydroorotate dehydrogenase (DHODH) and oxidized coenzyme Q10 (CoQ10). Fluorescence probes were used to observe the effect of DHODH inhibitor and CoQ10 on ROS level and Ca²＋concentration of endothelial cells. Results After DS treatment, the mitochondria showed pyknosis and mitochondrial volume significantly decreased, electron density of the mitochondrial membrane increased, and the number of cristae decreased in endothelial cells; the expression of cristae homeostasis related proteins MIC60 decreased by 23%, while cellular ROS level and Ca²＋ concentration increased; mitochondrial membrane potential decreased (P＜0.05 or P＜0.01). After GPR91 agonist treatment, the expression of cristae homeostasis related proteins MIC60 decreased by 31%, meanwhile, cellular ROS level increased by 27% and Ca²＋ concentration increased by 36%; mitochondrial membrane potential decreased (P＜0.05 or P＜0.01). After GPR91 inhibitor treatment, the expression of cristae homeostasis related proteins MIC60 increased by 22% and ATP5I increased by 40%; the levels of ROS decreased by 41% and Ca²＋ concentration decreased by 67%; and the mitochondrial membrane potential was restored to normal (P＜0.05 or P＜0.01). After DS treatment, the expression of DHODH decreased by 43% and the level of oxidized CoQ10 increased by 120% (P＜0.05 or P＜0.01). After GPR91 agonist treatment, the expression of DHODH decreased by 22% and the level of oxidized CoQ10 increased by 36% (P＜0.05 or P＜0.01). After GPR91 inhibitor treatment, the expression of DHODH increased by 40% and the level of oxidized CoQ10 decreased by 39% (P＜0.01). After DHODH inhibitor treatment, the ROS level increased by 20% and Ca²＋ concentration increased by 28%, and mitochondrial membrane potential reduced at same time (P＜0.05 or P＜0.01). Exogenous oxidized CoQ10 inhibited ROS production by 30% and decreased Ca²＋ concentration by 20% (P＜0.05 or P＜0.01). Conclusion Succinate/GPR91 promotes mitochondrial damage in endothelial cells, and its mechanism may relate to down-regulating the expression of DHODH and inhibiting the reduction of CoQ10 by affecting the mitochondrial cristae homeostasis.