Our previous studies have demonstrated that Jian-Pi-Yi-Shen formula (JPYSF), a traditional Chinese herbal decoction, has a renoprotective effect in 5/6 nephrectomy-induced chronic kidney injury. However, the role and potential mechanisms of JPYSF in the treatment of acute kidney injury (AKI) remain unknown. This study was designed to test the beneficial effect of JPYSF in an AKI mouse model and to investigate the underlying mechanism by using metabolomics analysis. The AKI mouse model was induced by a single intraperitoneal injection of cisplatin at a dose of 20 mg/kg. The mice in the treatment group were pretreated orally with JPYSF (18.35 g/kg/d) for 5 days before cisplatin injection. Seventy-two hours after cisplatin injection, serum and kidney samples were collected for biochemical and histological examination. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) was applied to analyze metabolic profiling variations in the kidney. The results showed that pretreatment with JPYSF obviously reduced the levels of serum creatinine and blood urea nitrogen and alleviated renal pathological injury in AKI mice. Orthogonal partial least-squares discriminant analysis (OPLS-DA) score plot revealed a clear separation between the AKI and AKI + JPYSF group. A total of 68 and 87 significantly differentially expressed metabolites were identified in the kidney of AKI mice responding to JPYSF treatment in negative and positive ion mode, respectively. The pivotal pathways affected by JPYSF included vitamin B6 metabolism, alanine, aspartate and glutamate metabolism, lysine biosynthesis, and butanoate metabolism. In conclusion, JPYSF can protect the kidney from cisplatin-induced AKI, which may be associated with regulating renal metabolic disorders. Copyright © 2020 Yuyan Li et al.