Vanadium compounds have been believed to be ideal drugs for diabetes biological therapy in future, but they suffer setback for the potential toxicity now. Toxicity study is necessary for vanadyl drugs development. This paper investigated the toxicity effects of vanadyl sulfate (VOSO4) oral administration in male Wistar rats using 1H NMR-based metabonomic analysis and clinical biochemical analysis. Rat urine were collected and their 1H NMR spectra were acquired, and then subjected to multi-variable statistical analysis. Compared to control groups, urinary excretion of lactate, TMAO, creatinine, taurine and hippurate increased following VOSO4 dosing, with concomitant decrease in the level of acetate and succinate. The dosed groups can be readily discriminated from the control groups by principle component analysis. The results showed that VOSO4 can affect energy metabolism process, interrupted intestinal microfloral metabolism, and induced liver and kidney injury. NMR-based metabonomic can offer additional information to traditional clinical chemistry in the sensitivity and specificity of results obtained.