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Gaillardin (GLN) is a sesquiterpene lactone isolated from the chloroform extract of Inula oculus-christi L. This natural compound has shown cytotoxicity in various cancerous cell line. However, its effect on leukemic cells is ambiguous. Due to the neurotoxicity of vincristine (VCR) in acute lymphoblastic leukemia (ALL), we aimed to examine the cytotoxic effects of GLN alone and in combination with vincristine on induction of apoptosis, and cell cycle progression in ALL cell lines (NALM-6 and MOLT-4). Our results displayed that GLN could induce cytotoxic effects in MOLT-4 and NALM-6 with IC50 values of 7.3 and 6.1 μM, respectively. In this study, we demonstrated that GLN induces cytotoxicity through G0/G1 phase arrest followed by apoptosis in a dose-dependent manner. Fortunately, this natural compound did not show significant cytotoxic effects on normal cells. This study demonstrated that GLN was capable to extend chemotherapeutic sensitivity in ALL cells by reducing VCR concentration without constraining its effectiveness. Therefore, it might act as a promising anticancer agent for the treatment of leukemia. [ABSTRACT FROM AUTHOR]