Autocrine motility factor (AMF) is well known for its ability to promote the motility, proliferation, and metastasis of cancer cells. However, extracellular AMF has been found to selectively kill cancer cells in an AMF type- and dose-dependent manner. In this study, the effects of eight different AMFs on HL-60 human leukemic cells were investigated. The AMF derived from HepG2 liver cancer cells (HG:AMF) was found to be more aggressive than the other AMFs and was further investigated for its inhibition of HL-60 cells in a dose- and time-dependent manner. Prolonged treatment with higher concentrations of HG:AMF was found to be severely detrimental to HL-60 cells. The inhibition of HL-60 cells by HG:AMF was visible under the microscope, as formazan deposits appeared in the sphere of aggregated HL-60 cells. HG:AMF was also found to significantly enhance ERK1/2 phosphorylation in a dose-dependent manner, while upregulating pro-apoptotic Bax and cleaved caspase 3 expression. No change was detected in the level of phosphorylated AKT and p70S6K, as well as anti-apoptotic Bcl-2. In a combination study, resveratrol (RV) was found to exert a cooperative and synergistic effect against the growth of HL-60 cells, while downregulating AKT activation. Cooperation with tamoxifen, a selective estrogen receptor modulator, was also suggested. Thus, HG:AMF alone or in combination with RV is recommended as a promising option for human leukemia therapy with the least risk of generating resistance.