Aims: Alantolactone (ALT) is active component of natural product Inula helenium with a lot of pharmacological effects, including anti-tumor effect. The present work aimed to explore the antitumor effect of ALT in B cell acute lymphoblastic leukemia (B-ALL). Main methods: B-ALL cells were treated with various concentrations of ALT, and then trypan blue assay, Annexin V/PI staining assay, PI staining assay, western blot analysis were employed to measure the effect of ALT on viability, apoptosis and cell cycle in B-ALL cells. In addition, a synthetic bioinformatics method was used to predict the underlying mechanism of antitumor effect of ALT. Then Reactive Oxygen Species (ROS) probe Dihydroethidium (DHE) and 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) were used to detect accumulation of cellular ROS. Meanwhile, DNA damage was identified by 8-oxoG, p-ATM1987, gamma-H2AX and comet assay. In addition, activity of glutathione reductase (GR), thioredoxin reductase (TrxR) and catalase were measured and overexpressed in SEM and RS4;11 cells to study the inhibition on these enzymes. Finally, B-ALL NOD-SCID mouse model was used to test its performance in vivo. Key findings: ALT showed good antitumor effect in B-ALL in vivo and in vitro through inducing ROS overload, which led to DNA damage. In addition, we found ROS overload caused by ALT was due to its direct inhibition on reductase. Significance: We found that ALT, a natural product, showing a promising tactic in the therapy of B-ALL by targeting ROS pathway.