Background: Acute myeloid leukaemia (AML) has a very high morbidity and mortality rate worldwide. The Chinese giant toad is used in traditional Chinese medicine (TCM) for the treatment of cancer, with bufalin being the main active ingredient. Hypothesis/Purpose: Bufalin treats AML by targeting autophagy to activate the atypical HIPPO pathway. Study Design: A network pharmacology and in vitro study approach was used to investigate the mechanism of apoptosis and autophagy in the treatment of AML with the TCM bufalin. Methods: We used network pharmacology to screen potential targets of bufalin in the regulation of AML, autophagy, and apoptosis, and validated the results through in vitro studies. Results: In web-based pharmacology, we selected the atypical HIPPO pathway. In vitro results showed that bufalin inhibited the proliferation of K562 cells and induced apoptosis and cell cycle arrest at the S phase, significantly increasing the level of autophagy in AML cells and releasing phosphorylated YAP1 to activate the atypical HIPPO pathway. Conclusion: The in vitro studies have demonstrated that bufalin can be regarded as a natural candidate with anticancer activity that regulates both autophagy and apoptosis. However, further in vivo and clinical studies are required to confirm its potential.