An apolipoprotein A-I mimetic peptide, D-4F, has been shown to improve vasodilation and inhibit atherosclerosis in hypercholesterolemic low-density lipoprotein receptor-null (LDLr-/-) mice. To study the metabolic variations of D-4F ininhibiting atherosclerosis, metabonomics, a novel system biological strategy to investigate the pathogenesis, was developed. Female LDLr-/- mice were fed a Western diet and injected with or without D-4F intraperitoneally. Atherosclerotic lesion formation was measured, whereas plasma metabolic profiling was obtained on the basis of ultra-high-performance liquid chromatography in tandem with time-of-flight mass spectrometry operating in both positive and negative ion modes. Data were processed by multivariate statistical analysis to graphically demonstrate metabolic changes. The partial least-squares discriminate analysis model was validated with cross-validation and permutation tests to ensure the model's reliability. D-4F significantly inhibited the formation of atherosclerosis in a time-dependent manner. The metabolic profiling was altered dramatically in hypercholesterolemic LDLr-/- mice, and a significant metabolic profiling change in response to D-4F treatment was observed in both positive and negative ion modes. Thirty-six significantly changed metabolites were identified as potential biomarkers. A series of phospholipid metabolites, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE), phosphatidylcholine (PC), phatidylethanolamine (PE), sphingomyelin (SM), and diacylglycerol (DG), particularly the long-chain LysoPC, was elevated dramatically in hypercholesterolemic LDLr-/- mice but reduced by D-4F in a time-dependent manner. Quantitative analysis of LysoPC, LysoPE, PC, and DG using HPLC was chosen to validate the variation of these potential biomarkers, and the results were consistent with the metabonomics findings. Our findings demonstrated that D-4F may inhibit atherosclerosis by regulating phospholipid metabolites specifically by decreasing plasma long-chain LysoPC.