Aim To investigate the effect of membrane-bound O-acyltransferase domain-containing 2 (MBOAT2) on mitophagy in vascular endothelial cells and its mechanisms. Methods Western blot was used to detect MBOAT2 protein expression in human atherosclerotic plaques. A human umbilical vein endothelial cell model with MBOAT2 overexpression was established. Western blot, transmission electron microscopy, JC-1 staining, MitoSOX Red staining, mRFP-GFP-LC3 dual-fluorescence staining and immunofluorescence staining were employed to assess the effects of MBOAT2 overexpression on mitochondrial injury, mitophagy, cytosolic mtDNA release, and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. In addition, the cGAS specific inhibitor RU.521 was used for intervention to verify the role of the cGAS-STING signaling pathway in mitochondrial injury and mitophagy dysfuntion. Results Compared with the control group, the expression of MBOAT2 protein in human atherosclerotic plaques increased by 70.7% (P＜0.05). After overexpression of MBOAT2, the expression levels of MIC60, ATP5L and ATP5I in human umbilical vein endothelial cells were downregulated by 15.6%, 18.9% and 25.8%, respectively (P＜0.05 or P＜0.01), accompanied by abnormal mitochondrial ultrastructure, decreased membrane potential and increased mitochondrial reactive oxygen species (mtROS); the expression levels of PINK1, Parkin, LC3Ⅱ and p62 proteins increased by 53.7%, 53.8%, 54.9% and 112.1%, respectively (P＜0.05 or P＜0.01), and the number of yellow autophagosomes and red autolysosomes increased; in addition, the expression level of cGAS protein increased by 121%, the phosphorylation level of TANK-binding kinase 1 (TBK1) increased by 52.5% (P＜0.05 or P＜0.01), and the release of mtDNA into the cytoplasm was observed. Compared with the MBOAT2 overexpression group, after RU.521 intervention, the expression level of cGAS protein decreased by 58.4% and the phosphorylation level of TBK1 decreased by 64.7%; PINK1, Parkin, LC3Ⅱ/LC3Ⅰ ratio and p62 decreased by 44.1%, 53.4%, 44.7% and 65.0%, respectively (P＜0.05 or P＜0.01). At the same time, the number of yellow autophagosomes and red autolysosomes decreased, the level of mtROS decreased, and the damage to mitochondrial cristae structure was alleviated. Conclusion MBOAT2 induces mitophagy dysfunction in vascular endothelial cells, thereby activating the mtDNA-cGAS-STING signaling pathway.