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Captive animal urine is rich in metabolites which is an important biological material for determining its estrus, pregnancy, health status, stress level and animal welfare by means of metabolite technique. However, urine samples are difficult to collect directly from the animal due to safety considerations. Urine samples were usually collected from the floor of animal’s enclosure after the animal’s excretion and moved to other place of the enclosure. Those samples are often contaminated by bacteria, which in turn affect the results of metabolite determination. It is necessary to explore a low-polluting and convenient urine collection and pretreatment method. In this paper, we collected 27 urine samples from eight captive giant pandas(Ailuropoda melanoleuca) (3 ♀, 5 ) by traditional methods, then each sample was divided into three groups for the control, filtrating with 0.22 μm filtration membrane and adding sodium azide treatments, respectively. Next, we used microbial cultures and Nuclear Magnetic Resonance (NMR) analysis and metabolite analysis to assay the bacteria removal efficacy of three treatments. The results showed that no bacteria were cultured in urine samples treated by filtration membrane, and there were no significant differences in the metabolites of NMR among three treatments. Further analysis found that seven and eight urinary metabolites significantly differed in genders and differed among age groups(both p < 0.05), respectively. The results suggest that the filter membrane pretreatment is effective for removing bacteria from urine samples, and can be used as a standard method for urine sample pretreatment and further evaluation of physiological indexes of giant pandas in metabolomics. Our findings not only enrich the basic metabolism database of giant pandas, and also provide data support for the health status monitoring, estrus and stress level assay in giant pandas and other captive wild animals. [ABSTRACT FROM AUTHOR]