Abstract: This study aimed to address the protective role of Guilingji (GLJ) against hydrocortisone-induced Kidney-Yang deficiency syndrome in rats with metabolites in serum, and explore its regulative approaches. KidneyYang deficiency syndrome rat model was constructed by high-dose injection of hydrocortisone. Rats were randomly divided into 6 groups:control group, model group, positive (Jinkui Shenqi Wan) group and low, medium, or highdose group of GLJ for continuous administration over 30 days. The efficacy of GLJ was evaluated with traditional pharmacodynamic indicators (body weight, behavioral indicators, and biochemical parameters) after the model was replicated successfully. Animal experimentation was approved according to the Committee on the Ethics of Animal Experiments of Shanxi University. Serum metabolic profiles obtained by UHPLC-Q Exactive Orbitrap-MS were used to explore metabolic regulation mechanism of GLJ. The results showed that GLJ could significantly improve Kidney-Yang deficiency syndrome. Pathway analysis showed that leucine-isoleucine metabolism, ether ester metabolism, and bile acid metabolism were the main pathways, with the main mechanism of action involving energy balance, intestinal homeostasis and immune function.