We present a metabolism-driven top-down systems biology approach to characterize metabolic changes in the mouse resulting from an infection with Plasmodium berghei, using high-resolution (1)H NMR spectroscopy and multivariate data analysis techniques. Twelve female NMRI mice were infected intravenously with approximately 20 million P. berghei-parasitized erythrocytes. Urine and plasma samples were collected 4-6 h before infection, and at days 1, 2, 3, and 4 postinfection. Multivariate analysis of spectral data showed differentiation between samples collected before and after infection, with growing metabolic distinction as the time postinfection progressed. Our analysis of plasma from P. berghei-infected mice showed marked increases in lactate and pyruvate levels, and decreased glucose, creatine, and glycerophosphoryl choline compared with preinfection, indicating glycolytic upregulation, and increased energy demand due to P. berghei infection. The dominant changes in the urinary metabolite profiles included increased levels of pipecolic acid, phenylacetylglycine, and dimethylamine, and decreased concentrations of taurine and trimethylamine- N-oxide, which may, among other factors, indicate a disturbance of the gut microbial community caused by the parasite. Although several of the observed metabolic changes are also associated with other parasitic infections, the combination of metabolic changes and, in particular, the occurrence of pipecolic acid in mouse urine postinfection are unique to a P. berghei infection. Hence, metabolic profiling may provide a sensitive diagnostic tool of Plasmodium infection and the control of malaria more generally.