Abstract: The metabonomic groups of male and female rat urine have been investigated by nuclear magnetic resonance (NMR) spectroscopy. Two pattern recognition techniques including the principal component analysis (PCA) and the orthogonal to partial least squares discriminant analysis (OPLS-DA) were used to find the difference of metabolic groups between male and female rats, respectively. The results of PCA and OPLS-DA analyses indicated that the amounts of valine, alanine, acetate, thiamine, ornithine, aminohippurate, phenylethylamine and cytosine in the urine samples of female rats were obviously higher than in those of male rats, based on the sufficient statistical variation (p<0.05). On the other hand, the amounts of methylamine, dimethylamine, trimethylamine, creatinine, allantoin, fumarate, formate in the urine samples of male rats were higher than those of female rats (p<0.05). The results showed that the gender of rat has certain effects on the metabolites of rat urine.