Atherosclerosis is a leading cause of morbidity and mortality in the Western countries. Mounting evidence points to the role of mitochondrial dysfunction in the pathogenesis of atherosclerosis. Recently, it has been shown that mitochondrial hydrogen sulfide (H₂S) can complement the bioenergetic role of Krebs cycle leading to improved mitochondrial function. However, controlled, direct delivery of H₂S to mitochondria was not investigated as a therapeutic strategy in atherosclerosis. Therefore, the aim of our study was to comprehensively evaluate the influence of prolonged treatment with mitochondrial H₂S donor AP39 on the development of atherosclerotic lesions in apolipoprotein E knockout (apoE^-/-) mice. Our results indicated that AP39 reduced atherosclerosis in apoE^-/- mice and stabilized atherosclerotic lesions through decreased total macrophage content and increased collagen depositions. Moreover, AP39 reduced proinflammatory M1-like macrophages and increased anti-inflammatory M2-like macrophages in atherosclerotic lesions. It also upregulated pathways related to mitochondrial function, such as cellular respiration, fatty acid β-oxidation and thermogenesis while downregulated pathways associated with immune system, platelet aggregation and complement and coagulation cascades in the aorta. Furthermore, treatment with AP39 increased the expression of mitochondrial brown fat uncoupling protein 1 (UCP1) in vascular smooth muscle cells (VSMCs) in atherosclerotic lesions and upregulated mRNA expression of other thermogenesis-related genes in the aorta but not perivascular adipose tissue (PVAT) of apoE^-/- mice. Finally, AP39 treatment decreased markers of activated endothelium and increased endothelial nitric oxide synthase (eNOS) expression and activation. Taken together, mitochondrial H₂S donor AP39 could provide potentially a novel therapeutic approach to the treatment/prevention of atherosclerosis.