Abstract Britannin, a Sesquiterpene Lactone isolated from Inula aucheriana, has recently gained attractions in the therapeutic fields due to its vast cytotoxic properties in different types of cancers. This study was designed to evaluate the cytotoxic effect of this agent on Acute Lymphoblastic Leukemia (ALL) cell lines, either as a monotherapy or in combination with Vincristine (VCR). The results obtained in this study showed that while Britannin reduced the viability of ALL cell lines such as NALM-6, REH, and JURKAT cells, it did not exert cytotoxicity against normal Peripheral Blood Mononuclear Cells (PBMCs) and L929 cells. Among tested cells, pre-B ALL-derived NALM-6 cells had the highest sensitivity to Britannin. Moreover, we found that Britannin induced p21/p27-mediated G1 cell cycle arrest cells and Reactive Oxygen Specious (ROS)-mediated apoptotic cell death in NALM-6 cells. When NALM-6 cells were treated with N-acetyl-L-Cysteine (NAC), a scavenger of ROS, we found that Britannin could induce neither apoptosis nor reduce the survival of the cells, suggesting that the cytotoxic effect of Britannin is induced through ROS-dependent manner. The cytotoxic effect of Britannin also was potentiated by autophagy suppression using Chloroquine (CQ). Moreover, we found that a low dose of Britannin enhanced the effect of Vincristine in NALM-6 cells by inducing apoptotic cell death via altering the expression of apoptotic-related genes. Overall, our results proposed a mechanism for the cytotoxic effect of Britannin, either as a single agent or in combination with Vincristine, in NALM-6 cells.