Drug-mediated or medical condition-mediated disruption of hERG function accounts for the main cause of acquired long-QT syndrome (acLQTs), which predisposes affected individuals to ventricular arrhythmias (VA) and sudden death. Many Chinese herbal medicines, especially alkaloids, have risks of arrhythmia in clinical application. The characterized mechanisms behind this adverse effect are frequently associated with inhibition of cardiac hERG channels. The present study aimed to assess the potent effect of Rutaecarpine (Rut) on hERG channels. hERG-HEK293 cell was applied for evaluating the effect of Rut on hERG channels and the underlying mechanism. hERG current (I-hERG) was measured by patch-clamp technique. Protein levels were analysed by Western blot, and the phosphorylation of Sp1 was determined by immunoprecipitation. Optical mapping and programmed electrical stimulation were used to evaluate cardiac electrophysiological activities, such as APD, QT/QTc, occurrence of arrhythmia, phase singularities (PSs), and dominant frequency (DF). Our results demonstrated that Rut reduced the I-hERG by binding to F656 and Y652 amino acid residues of hERG channel instantaneously, subsequently accelerating the channel inactivation, and being trapped in the channel. The level of hERG channels was reduced by incubating with Rut for 24 hours, and Sp1 in nucleus was inhibited simultaneously. Mechanismly, Rut reduced threonine (Thr)/ tyrosine (Tyr) phosphorylation of Sp1 through PI3K/Akt pathway to regulate hERG channels expression. Cell-based model unables to fully reveal the pathological process of arrhythmia. In vivo study, we found that Rut prolonged QT/QTc intervals and increased induction rate of ventricular fibrillation (VF) in guinea pig heart after being dosed Rut for 2 weeks. The critical reasons led to increased incidence of arrhythmias eventually were prolonged APD(90) and APD(50) and the increase of DF, numbers of PSs, incidence of early after-depolarizations (EADs). Collectively, the results of this study suggest that Rut could reduce the I-hERG by binding to hERG channels through F656 and Y652 instantaneously. While, the PI3K/Akt/Sp1 axis may play an essential role in the regulation of hERG channels, from the perspective of the long-term effects of Rut (incubating for 24 hours). Importantly, the changes of electrophysiological properties by Rut were the main cause of VA.