Tripterygium wilfordii Hook. F. is a plant used in traditional Chinese medicine to treat rheumatoid arthritis, lupus erythematosus, and psoriasis in China. However, its main active substance, triptolide, has toxic effects on the heart, liver, and kidneys, which limit its clinical application. Therefore, determining the mechanism of cardiotoxicity in triptolide and identifying effective early-warning biomarkers is beneficial for preventing irreversible myocardial injury. We observed changes in microRNAs and aryl hydrocarbon receptor (AhR) as potential biomarkers in triptolide-induced acute cardiotoxicity by using techniques such as polymerase chain reaction (PCR) assay. The results revealed that triptolide increased the heart/body ratio and caused myocardial fiber breakage, cardiomyocyte hypertrophy, increased cell gaps, and nuclear dissolution in treated male rats. Real-time PCR array detection revealed a more than 2-fold increase in the expression of 108 microRNA genes in the hearts of the male rats; this not only regulated the signaling pathways of ErbB, FOXO, AMPK, Hippo, HIF-1α, mTOR, and PI3K-Akt but also participated in biological processes such as cell adhesion, cell cycling, action potential, locomotory behavior, apoptosis, and DNA binding. Moreover, triptolide reduced the circulatory and cardiac levels of AhR protein as a target of these microRNAs and the messenger RNA expression of its downstream gene CYP1 A1. However, decreases in myocardial lactate dehydrogenase, creatine kinase MB, catalase, and glutathione peroxidase activity and an increase in circulating cardiac troponin I were observed only in male rats. Moreover, plasma microRNAs exhibited dynamic change. These results revealed that circulating microRNAs and AhR protein are potentially early-warning biomarkers for triptolide-induced cardiotoxicity.