BACKGROUND Although great progress has been made in the pathogenesis and treatment of acute kidney injury (AKI), it still has high incidence and poor prognosis. The present study was performed in order to further understand the metabolomic changes of ischemia/reperfusion (I/R)-induced AKI and the protective effect of L-carnitine on AKI. METHODS Kidney tissues and serum samples were collected at different time points from three groups of rats including control group, I/R group and L-carnitine-pretreated group. High-performance liquid chromatography coupled with mass spectrometry-based metabolomics approach was applied to investigate the characteristic of I/R-induced AKI and the protective effects of L-carnitine in rat kidney I/R model. Antioxidant enzymatic activity and phospholipase A(2) activity were determined to validate the metabolic outcomes. RESULTS Changes in the pattern of endogenous metabolites as a result of kidney I/R injury were readily detected as early as 2 h after reperfusion, and earlier than the increase in blood urea nitrogen and serum creatinine. Twenty-eight differential endogenous metabolites were discovered and structurally identified by MS(n) analysis. After I/R injury, lysophospholipids, free fatty acids and nitrotyrosine significantly increased, while carnitine and acetyl-carnitine significantly decreased compared to control. Phospholipase A(2) activity and malondialdehyde level also increased, while superoxide dismutase activity decreased in kidney I/R injury rats. Treatment of L-carnitine 30 min prior to reperfusion significantly relieved I/R-induced metabolomic changes. CONCLUSIONS I/R-induced AKI could be characterized by oxidative stress and changes in lipid metabolism through metabolomic investigation, and L-carnitine treatment 30 min before reperfusion had protective effects against I/R-induced AKI.