Enhanced green fluorescent protein (EGFP) is a widely used biological reporter. However, the effects of EGFP expression in vivo are still unclear. To investigate the effects of EGFP transgenic expression in vivo, we employed an NMR-based metabonomics method to analyze the metabonome of EGFP transgenic mice. The results show that the metabonomes of urine, liver, and kidney of the EGFP transgenic mice are different from their wild-type counterparts. The EGFP mice expressed high levels of urinary 3-ureidopropionate, which is due to the down-regulated transcriptional level of β-ureidopropionase. The expression of EGFP in vivo also affects other metabolic pathways, including nucleic acid metabolism, energy utilization, and amino acids catabolism. These findings indicate that EGFP transgenic expression is not as inert as has been considered. Our investigation provides a holistic view on the effect of EGFP expression in vivo, which is useful when EGFP is employed as a functional biological indicator. Our work also highlights the potential of a metabonomics strategy in studying the association between molecular phenotypes and gene function.