Ethnopharmacological relevance: Traditional Chinese medicinal herb Salvia miltiorrhiza Bunge(Danshen) and its components have been widely used to treat cardiovascular diseases for hundreds of years in China, including hypertension, diabetes, atherosclerosis, and chronic heart failure. Salvia miltiorrhiza injection (SMI), an aqueous extracts of Salvia miltiorrhiza Bunge, is one of most widely used traditional Chinese medicine injections. SMI is widely used in the treatment of diabetic vascular complications, However, the mechanisms remain to be defined. Aim of the study: To investigate protective mechanism of Salvia miltiorrhiza Bunge against ROS generation in VSMCs of diabetic mice and patients. Materials and methods: Salvia miltiorrhiza injection (hereinafter referred to as SMI, 1.5 g mL(-1)), which was approved by the State Food and Drug Administration (approval number: Z32020161), was obtained from Shenlong Pharmaceutical Co., Ltd. (batch number: 11040314). SMI or vehicle were intraperitoneally administrated to the HFD-fed db/db mice, artery was harvested after 24weeks later. qRT-PCR and Western blot analysis were used to detect the expression of KLF6, KLF5, KLF4, KLF10, KLF12, and HO-1. DCFH-DA staining detected intracellular ROS production. Loss- and gain-of-function experiments of KLF10 were used to investigate the effect of KLF10 on the expression of HO-1. Dual-luciferase reporter assay evaluated the effect of KLF10 on the activity of the HO-1 promoter. Results: KLF10 expression and ROS generation are significantly increased in the arteries of HFD-fed db/db mice, VSMCs of diabetic patients, as well as in high glucose-treated VSMCs. KLF10 overexpression suppresses, while its knockdown facilitates the expression of heme oxygenase (HO-1) mRNA and protein. Further, Salvia miltiorrhiza injection (SMI) abrogates KLF10 upregulation and reduces ROS generation induced by high glucose in VSMCs. Mechanistically, KLF10 negatively regulates the HO-1 gene transcription via directly binding to its promoter. Accordingly, SMI treatment of VSMCs reduces ROS generation through inhibiting KLF10 expression and thus relieving KLF10 repression of the expression of HO-1 gene, subsequently contributing to upregulation of HO-1. Conclusion: SMI exerts anti-oxidative effects on VSMCs exposed to high glucose through inhibiting KLF10 expression and thus upregulating HO-1.