Metabonomics technology was used to investigate the mechanism of Liuwei Dihuang Pill treating acute kidney injury induced by gentamicin.The ultra performance liquid chromatography-mass spectrometry(UPLC-MS) coupled with principle component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were used to evaluate the changes of biomarkers of control group,model group and treatment group.Significant differences between the control group,model group and treatment group were observed in clustering analysis,PCA and OPLS-DA model.Seventeen potential biomarkers between control group and model group were identified as multiple phosphatidycholines,sphinganine,phytosphingosine,SM(d18∶0/16∶1(9z)),arachidonic acid.The relative contents of sphinganine,phytosphingosine,SM(d18∶0/16∶1(9z)) and arachidonic acid increased in model group compared with control group,while decreased in treatment group.Therefore,Liuwei Dihuang Pill may achieve the aim to treat acute kidney injury induced by gentamicin through adjusting the content of these biomarkers.This study provides a theoretical basis for Liuwei Dihuang Pill treating kidney injury in clinical practice.