Steroids are potential anti-leukemia agents, and Epigynum auritum is a Yunnan folk medicine with high levels of androsterone, pregnane, and steroid derivatives. However, the underlying therapeutic mechanism of androsta4,6,8,14-tetraene-3,11,16-trione (ATT), an androsterone isolated from Epigynum auritum , is not yet clear. This study aimed to explore the anti-leukemia mechanism of ATT using molecular biology, network pharmacology, and molecular docking technology. The cell viability results showed that ATT had an anti-proliferation effect in acute lymphoblastic leukemia cells (CEM/C1, MOLT-4, Jurkat, BALL-1, Nalm-6, and RS4;11). Further studies showed that ATT reduced the mitochondrial membrane potential in B-cell acute lymphoblastic leukemia cell lines (BALL-1, Nalm-6, and RS4;11) and induced cell cycle arrest in MOLT-4 and BALL-1. ATT induced BALL-1 cell apoptosis by activating Caspase 3/7 activity and causing DNA fragmentation. Network pharmacology results suggested that ATT exerts its anti-leukemia activity via the PI3K/Akt signaling pathway. In addition, molecular docking analysis showed that ATT had high scores in docking with PTGS2, NR3C1, and AR. Western blotting results showed that ATT reduced the relative protein level of P-PI3K and P-Akt, thereby increasing the relative level of pro-apoptosis protein Bax and reducing the relative level of anti-apoptosis protein Bcl-2, the apoptosis downstream protein pro-caspase3, and cell proliferation-related proteins (P-GSK3B and CyclinD1). In conclusion, these results demonstrated that ATT could be a potential candidate drug with apoptosis-induction and cell cycle arrest effects for further investigation in acute lymphoblastic leukemia therapy.