Background: Xixin has been widely used as a traditional Chinese medicine for headache, toothache and inflammatory diseases. Clinical investigation indicated that adverse drug reactions occurred with an overdose of xixin, but the toxic mechanism of xixin in vivo based on trace elements has not been researched yet. Objective: To explore the in vivo toxic mechanism of xixin induced by trace elements. Materials and Methods: The contents of trace elements in the serum and liver of mice were determined by inductively coupled plasma-mass spectrometry (ICP-MS) after obtaining xixin extracts. Principal component analysis (PCA) and cluster analysis (CA) were performed between the trace elements' content and dosage using the software GeneSpring 12.1 to analyze the main toxic elements in vivo. Results: Trace elements' contents were obviously raised after xixin extracts were taken as a dosage of 150 mg/mL and 50 mg/mL, respectively. Na, Ca, Cu and Cd in serum and Ca and Zn in liver were the main trace elements inducing the toxic reaction of xixin. Conclusion: Xixin possesses the potential function of indirectly upregulating trace elements in vivo. This study, for the first time, elucidated the in vivo toxic mechanism of xixin based on trace elements. This method could also be utilized in the research of corresponding aspects.