Background and Purpose: Nitidine chloride (NC) is a standard active component of a traditional Chinese medicine Zanthoxylum nitidum (Roxb.) DC. (ZN). NC has certain toxic effects which make it a potential anti-tumor monomer. However, the cardiotoxicity of anti-tumor drugs has been a concern, the cardiotoxicity of NC has not been reported. Thus, we aimed to investigate the cardiotoxicity of NC and elucidate its mechanism of action. Experimental Approach: The aqueous extract of ZN was administered intraperitoneally to rats, and NC was similarly administered to beagles and mice once daily for 4 weeks. Cardiac function was assessed by echocardiography while NC-induced changes in cardiac signaling pathways were investigated using proteomics and western blot. To investigate the mechanism of NC-induced toxicity, adeno-associated virus 9-autophagy-related 4B cysteine peptidase (ATG4B) was injected into mice. Corresponding in vitro experiments were performed. Key Results: ZN and NC induced cardiac hypertrophy in rats and mice, respectively. Beagle death resulted from NC was mediated by NC-induced changes to mitochondrial components and peptidase activity of the heart. Consistently, we observed reduced cardiac autophagy levels in NC-treated mice. Specifically, we found that ATG4B may be a potential target of NC as the overexpression of ATG4B reversed cardiac hypertrophy and reduced autophagy levels observed in NC-treated mice. Conclusion and Implications: ZN resulted in cardiac hypertrophy in rats. NC induced cardiac hypertrophy via ATG4B-mediated downregulation of autophagy in mice. Thus, this study provides guidance for the safe clinical application of ZN and the use of NC as an anti-tumor drug.