Huangqi-Danshen decoction (HDD) is composed of Astragali Radix (Huang-qi) and Salviae Miltiorrhizae Radix et Rhizoma (Dan-shen), both of which are the most commonly used herbs for the clinical treatment of diabetic nephropathy (DN) in traditional Chinese medicine and show good efficacy. However, the underlying mechanism of this effect is unclear. The aim of this study was to evaluate the effect and potential mechanism of HDD in the treatment of DN in a type 2 diabetic animal model, db/db mice. HDD extract was administered orally to db/db mice at a dose of 6.8 g/kg/day for 12 weeks. At the end of the study, serum, urine, and kidney samples were collected for biochemical and pathological examination. The expression of proteins associated with mitochondrial fission and mitophagy was determined by quantitative real-time PCR, Western blotting, and immunohistochemical analysis. The results showed that treatment with HDD substantially reduced urinary albumin excretion and improved renal injury in db/db mice. Moreover, mitochondrial fission was increased in the kidneys of the db/db mice, as evidenced by enhanced expression of dynamin-related protein 1 and mitochondrial morphological changes. Furthermore, PTEN-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy was activated in the db/db mice, which manifested as increased protein expression and obvious autophagic vacuole encapsulating mitochondria. HDD treatment significantly reversed the enhanced mitochondrial fission and PINK1/Parkin-mediated mitophagy in the db/db mice. In conclusion, this work suggested that HDD could protect against type 2 diabetes-induced kidney injury possibly by inhibiting PINK1/Parkin-mediated mitophagy. AJTR Copyright © 2020.