Atherosclerosis (AS) is a chronic vascular disease primarily affecting large and medium-sized arteries and involves various complex pathological mechanisms and factors. Previous studies have demonstrated a close association between atherosclerosis and inflammatory damage, metabolic disorders, and gut microbiota. It is also closely linked to several cellular processes, such as endothelial cell pyroptosis, ferroptosis, mitophagy, mitochondrial dynamics, and mitochondrial biogenesis. Mitophagy has been recognized as a previously unexplored mechanism contributing to endothelial injury in atherosclerosis. Our study aims to further elucidate the potential relationship and mechanisms between AS-induced mitophagy dysfunction and the interaction of TMBIM6 and NDUFS4. Data from the study demonstrated that atherosclerosis in AS mice was associated with substantial activation of inflammatory and oxidative stress damage, along with a marked reduction in endothelial mitophagy expression and increased pathological mitochondrial fission, leading to mitochondrial homeostasis disruption. However, under pharmacological intervention, mitophagy levels significantly increased, pathological mitochondrial fission was notably reduced, and oxidative stress and inflammatory damage were suppressed, while necroptotic pathways in endothelial cells were significantly blocked. Interestingly, the deletion of TMBIM6 or NDUFS4 in animal models or cell lines markedly impaired the therapeutic effects of the drug, disrupting its regulation of mitophagy and mitochondrial fission, and leading to the re-emergence of inflammatory responses and oxidative stress damage. Metabolomics analysis further revealed that autophagy plays a pivotal regulatory role during drug intervention and after genetic modification of TMBIM6 and NDUFS4. The activation of autophagy (macroautophagy/mitophagy) alleviated the negative effects of mitochondrial fission and inflammatory damage induced by lipid stress in endothelial cells, a regulatory mechanism likely associated with the TMBIM6-NDUFS4 axis. Subsequent animal gene modification experiments demonstrated that knocking out TMBIM6-NDUFS4 negates the therapeutic effects of the drug on lipid-induced damage and metabolic function. In summary, our research reveals a phenotypic regulatory mechanism of endothelial cell stress damage through mitophagy, influenced by the interaction of TMBIM6 and NDUFS4. Pharmacological intervention can restore mitochondrial homeostasis in endothelial cells by regulating mitophagy via the TMBIM6-NDUFS4 pathway. This novel insight suggests that TMBIM6-NDUFS4 may serve as a key therapeutic target for atherosclerosis.