Abstract: The Chinese herbal Radix Scrophulariae is the main medicine for nourishing yin and reducing fire. It can be used to treat hyperthyroidism due to yin deficiency and fire hyperactivity, but its mechanism is not clear. The present study was aimed to explore the mechanism of Radix Scrophulariae treatment of hyperthyroidism due to yin deficiency and fire hyperactivity. The urine metabolomic approach was conducted using the method of UPLC-TOF-MS. The results showed that Radix Scrophulariae has good therapeutic effects on hyperthyroidism rat model of yin deficiency. After treatment with Radix Scrophulariae, through metabolic profiling and protocol analysis, 6 potential metabolic markers may be closely related with the treatment mechanism of Radix Scrophulariae on this disease, including proline betaine, estrone, thymidine, 5-hydroxyindoleacetic acid, cyclic AMP and L-dopa. The strongest metabolic pathways were associated with tryptophan metabolism, pyrimidine metabolism, tyrosine metabolism, purine metabolism and steroid hormone biosynthesis. The urine metabolomic approach can be applied to clarify the therapeutic mechanism of Radix Scrophulariae on hyperthyroidism rat of yin deficiency, and provide the theoretical basis for the clinical practice of Radix Scrophulariae on nourishing yin to reduce pathogenic fire.