Hexamethylene bisacetamide (HMBA) and natural flavanoid baicalin both exert potent antileukemic activity. However, there is currently no data on the anti-leukemic effects of baicalin in combination with HMBA. In the present study, we demonstrated that the combination of baicalin and HMBA synergistically inhibited the proliferation of acute myeloid leukemia (AML) cell lines. In addition, a slight G0/G1 phase arrest and significant apoptosis were observed. The combination treatment triggered apoptosis through the intrinsic pathway, which involved loss of MMP, decreased Bcl‑2/Bax ratio and Bcl‑XL/Bax ratio, caspase‑9 activation, as well as through the extrinsic pathway mediated by Fas and caspase‑8 activation. On the other hand, combination of baicalin and HMBA showed little toxic effect on peripheral blood mononuclear cells from healthy volunteers. Our results raise the possibility that the novel combination of baicalin and HMBA may be a promising regimen for the treatment of AML.