Benzene is known to produce hematotoxicity in occupational exposure workers. This study examined the utility of metabonomic biomarkers to ascertain subacute toxicity produced by benzene in male C3H/He mice. A 30-d intermittent collection of urine was obtained from mice in this experiment. The relative organ weights, blood parameters, and bone marrow smears were examined to identify specific changes of benzene-induced toxicity. In addition, an integrated analytical approach based on liquid chromatography coupled with mass spectrometry (LC-MS) was developed to map metabolic responses in urine. Five endogenous metabolites, hypoxanthine, spermidine, 4-aminohippuric acid, indolelactic acid, and glutamylphenylalanine, were identified as potential biomarkers of benzene-induced toxicity, indicating that pathways of purine, spermidine, fatty acid, tryptophan, and peptides metabolism might be disturbed in benzene-exposed mice. Our findings showed that the use of urine metabonomics was a more sensitive tool to detect benzene-induced toxicity compared to body weight or blood parameter changes.