Background: It has been demonstrated that scutellarin, a natural flavone compound from Scutellaria lateriflora and Scutellaria barbata, exerts selective cytotoxicity against a range of cancer cells. However, the underlining mechanism of scutellarin on acute myeloid leukemia (AML) remains elusive. Methods: In this study, the combination of network pharmacology and experimental verification was performed to identify the pharmacological mechanisms of scutellarin for AML therapy. The public databases, such as PharmMapper, UniProt, OMIM, GeneCards, DrugBank and PharmGkb database, were used to sceen the potential targets of scutellarin and AML. The protein-protein interaction (PPI), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted to uncover the mechanism of scutellarin in the treatment of AML. Finally, the network pharmacological results were further confirmed by in vitro and in vivo experiments. Results: First and foremost, we totally obtained 289 target genes for scutellarin and 10998 disease targets for AML. 253 overlapping genes were preliminarily considered the potential targets of scutellarin for AML treatment. The results of PPI network analysis, GO analysis and KEGG pathway enrichment demonstrated that the anti-AML effect of scutellarin may focused on MAPK signaling pathway. Furthermore, the cytologic tests suggested that scutellarin can inhibit AML cells proliferation through the mediation of JNK/Caspase-3 pathway. Meanwhile, pretreatment with the JNK inhibitor SP600125 rescued scutellarin-induced apoptosis. Similarly, scutellarin obviously suppressed subcutaneous xenograft growth in nude mice via regulating the JNK/Caspase-3 signaling pathway. Conclusion: In this study, we integrated network pharmacology-based prediction and experimental validation and revealed the importance of the JNK pathway in scutellarin-mediated AML treatment.