Objective To study metabonomics in the urine of rats of different genders by magnetic resonance (MR) with 2 normalization methods. Methods Different normalization methods such as mean-centering not scaling (Ctr) and unit variance scaling (UV) were used before orthogonal to partial least squares discriminant analysis(OPLS-DA). Distinguished metabolites in the urine of different gender rats were analyzed by calculating the correlation coefficients. Results The data normalized by Ctr before OPLS-DA analysis revealed high degree conception metabolites in the urine such as valine, alanine, acetate, ornithine, aminohippurate, phenylethylamine, cytosine, citrate, dimethylamine, allantoin, methylamine, fumarate and one unknown metabolite whose chemical shift was δ4.14. Data normalized by UV before OPLS-DA analysis revealed the above 12 high degree conception metabolites except citrate, and also low degree conception metabolites such as thiamine, creatinine, formate and one unknown metabolite whose chemical shift was δ2.92.Conclusion Unit variance scaling is a more effective normalization method in metabonomic analysis.