Diabetes mellitus (DM) is a chronic, noncommunicable disease, mainly due to insufficient insulin secretion or insulin sensitivity. Atherosclerosis is one of the major macrovascular complications of DM, and it is the main cause of death from clinical observation. Mongolian acupuncture therapy has the function of dredging meridians and regulating blood circulation, which has been proved to have unique efficacy and notable advantages in the treatment of DM and its common chronic complications. However, the effects and molecular mechanism of Mongolian acupuncture therapy in T2DM atherosclerosis remain unclear. The aim of this study is to explore the effect and molecular mechanism of streptozotocin and vitamin D3-induced T2DM atherosclerosis in rats, with or without Mongolian acupuncture therapy. The results of serum biochemical and histopathology analysis suggest that acupuncture therapy can improve cellular inflammatory factors, kidney function, serum lipid function and upper carotid artery tissue lesions in rat with T2DM atherosclerosis, which is an effective treatment method. In addition, we applied proteomics and metabolomics to comprehensively demonstrate that Mongolian acupuncture therapy in T2DM atherosclerosis mainly through regulation of lipid metabolism and amino acid metabolism. In general, our findings provide beneficial data support for future clinical studies of Mongolian acupuncture therapy in T2DM atherosclerosis.