After our serum metabonomic study of colorectal cancer (CRC) patients recently published in J. Proteome Res., we profiled urine metabolites from the same group of CRC patients (before and after surgical operation) and 63 age-matched healthy volunteers using gas chromatography-mass spectrometry (GC-MS) in conjunction with a multivariate statistics technique. A parallel metabonomic study on a 1,2-dimethylhydrazine (DMH)-treated Sprague-Dawley rat model was also performed to identify significantly altered metabolites associated with chemically induced precancerous colorectal lesion. The orthogonal partial least-squares-discriminant analysis (OPLS-DA) models of metabonomic results demonstrated good separations between CRC patients or DMH-induced model rats and their healthy counterparts. The significantly increased tryptophan metabolism, and disturbed tricarboxylic acid (TCA) cycle and the gut microflora metabolism were observed in both the CRC patients and the rat model. The urinary metabolite profile of postoperative CRC subjects altered significantly from that of the preoperative stage. The significantly down-regulated gut microflora metabolism and TCA cycle were observed in postoperative CRC subjects, presumably due to the colon flush involved in the surgical procedure and weakened physical conditions of the patients. The expression of 5-hydroxytryptophan significantly decreased in postsurgery samples, suggesting a recovered tryptophan metabolism toward healthy state. Abnormal histamine metabolism and glutamate metabolism were found only in the urine samples of CRC patients, and the abnormal polyamine metabolism was found only in the rat urine. This study assessed the important metabonomic variations in urine associated with CRC and, therefore, provided baseline information complementary to serum/plasma and tissue metabonomics for the complete elucidation of the underlying metabolic mechanisms of CRC.