Timosaponin AIII (TAIII) is a steroidal saponin isolated from Anemarrhena asphodeloides that has been shown to inhibit cell growth and induce apoptosis in cancer. However, the effect of TAIII on acute myeloid leukemia (AML) remains unclear. Here, the molecular mechanism by which TAIII-induced apoptosis affects human AML cells was investigated. The results showed that TAIII significantly inhibited cell proliferation of four AML cell lines (MV4-11, U937, THP-1, and HL-60). Furthermore, TAIII induced apoptosis of HL-60 cells through caspase-3, caspase-8, and caspase-9 activations and PARP cleavage in a dose- and time-dependent manner. Moreover, Western blot analysis also showed that TAIII increased phosphorylation of JNK1/2 and p38 MAPK in a dose-dependent manner. Inhibition of JNK1/2 by specific inhibitors significantly abolished the TAIII-induced activation of the caspase-8. Taken together, our results suggest that TAIII induces HL-60 cell apoptosis through JNK1/2 pathways and could serve as a potential additional chemotherapeutic agent for treating AML.