Acute Myeloid Leukemia (AML), a cancer of the myeloid line of blood cells, progresses rapidly and is typically fatal within weeks or months if left untreated. Asparaginases are a class of enzymatic anti-leukemia agents that induce apoptosis in leukemia cell lines; however, the role of L-asparaginase in the induction of apoptosis in AML cells has not been investigated. In this study, we investigated the apoptosis-inducing effect of L-asparaginase and its underlying mechanism in AML U937 cells. The results showed that L-asparaginase significantly inhibited the proliferation of U937 cells by inducing apoptosis. Furthermore, the low baseline expression level of asparaginase synthase (ASNS) demonstrated the sensitivity of U937 cells and AML M5, a rare subtype of AML, to L-asparaginase. Apoptosis induced by L-asparaginase is mediated by apoptosis-inducing factor (AIF). Our findings show the potential of L-asparaginase as an effective approach in treating AML via the induction of apoptosis mediated by AIF.